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Collagen fibril assembly during postnatal development and dysfunctional regulation in the lumican-deficient murine cornea
Developmental Dynamics
Abstract
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+/+) and lumican-null (Lum-/-) mice. In Lum+/+ 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-/- 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+/+ 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-/- 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.
... Chen et al. 2014), and decorin, fibromodulin and lumican play a role in tendon development, modulating fiber morphology, diameter and arrangement as well as interfibrillar space (Ezura et al. 2000)(G. Zhang et al. 2006). In vitro fibrillogenesis (turbidity) assays suggest that decorin, lumican and biglycan downregulate collagen gelation with a decreased fibril formation plateau and that their core proteins play distinct roles compared to intact SLRPs (with GAG side chains) (Rada, Cornuet, and Hassell 1993) (Reese, Underwood, and Weiss 2013). ...
... Chen et al. 2014) and tendon (G. Zhang et al. 2006) that require well-organized fibrous networks for their physiological functions. For example, lumican and biglycan deficiency cause an impaired lamellar organization of collagen fibers and damage the transparency of cornea (S. ...
... Chen et al. 2014), and decorin-, fibromodulin-and lumican-deficient mice show abnormal fiber morphology, altered fiber size distributions and atypically non-uniform interfibrillar space in tendon (Ezura et al. 2000)(G. Zhang et al. 2006 and demonstrated abnormal collagen fibril formation and irregular collagen structure in skin (Maccarana et al. 2009). Unlike aggrecan, which is predominantly expressed in cartilage, versican is more universally distributed throughout the human body and shows increased deposition and turnover during numerous fibroproliferative process (Theocharis 2008)(Bode- Lesniewska et al. 1996). ...
Type I collagen is the most abundant structural protein in the extracellular matrix (ECM), forming a dynamic 3D fibrous network that is highly regulated by other ECM components including proteoglycans (PGs) and glycosaminoglycans (GAGs). Matrix PGs, especially the small leucine rich PG (SLRP) subgroup, have been well studied as collagen binding proteins and regulators of fibrillogenesis. However, the impact of the hyalectan subgroup of PGs, particularly versican, on collagen behaviors is not well understood. There is a particular need for understanding the role of versican in the collagen network because of its universal distribution in tissues and its altered expression during collagen-related fibrotic disorders. My aim was to study collagen/versican interactions and to investigate the role of versican in modulating collagen structural and mechanical behaviors. I used solid phase binding assays and the Collagen Toolkit to identify binding sites, and I carried out in vitro turbidity assays combined with fibroblast-derived matrices (FDM) to study fibrillogenesis. Collagen fiber organization was visualized using scanning electron microscopy (SEM), and cell-mediated collagen realignments and contractions were assessed by collagen plug and engineered microtissue assays. Shear rheometry was carried out on collagen gels and liver tissues to evaluate the impact of versican on tissue mechanics. I determined that versican and its V3 isoform bind collagen via the versican G3 domain and collagen R-G-Hydrophobic-O motif, independent of versican GAG residues. Compared to SLRPs and the structurally similar hyalectan aggrecan, versican shows unique effects on multiple collagen behaviors: 1) versican upregulates collagen gelation and promotes the deposition of collagen-rich matrix with aligned fibers; 2) the presence of versican improves fibril fusion into large bundles and forms a looser network; 3) versican improves cell-mediated collagen compaction, alignment and microtissue contraction; 4) versican contributes to collagen gel mechanics by decreasing stiffness and attenuating strain stiffening. In tissues, versican and its GAGs also play a role by downregulating compression stiffening. Thus, versican is a unique regulator of various collagen behaviors and therefore has potential therapeutic value in collagen-related fibroproliferative diseases such as inflammation, fibrosis and cancer.
... These cellular protrusions/processes are distinct and common during the course of stromal development and particularly lamella formation (Birk and Trelstad, 1984;Young et al., 2014). Corneal development continues in the postnatal period, yielding complete transparency (Chakravarti et al., 2006;Coulombre and Coulmbre, 1958;Coulombre and Coulombre, 1958;Song et al., 2003). Analysis of stromal development in embryonic chickens demonstrated that collagen fibrillogenesis occurred within small surface recesses of the keratocytes (Birk and Trelstad, 1984). ...
... Class II SLRPs show both temporal and spatial differences. While lumican and keratocan are homogeneous across the cornea stroma at birth, the expression of lumican is restricted to the posterior stroma after maturation (Chakravarti et al., 2006;Chen et al., 2010;Zhang et al., 2009). Fibromodulin expression in the corneal stroma is strongest at P14, but is decreased and restricted to the peripheral cornea with maturation (Chen et al., 2010). ...
... Mouse models have demonstrated that SLRPs are critical regulators of collagen fibrillogenesis, particularly the linear and lateral growth of protofibrils into mature fibrils (Chakravarti et al., 1998;Chakravarti et al., 2003;Chakravarti et al., 2006;Chen et al., 2010;Ezura et al., 2000;Mienaltowski and Birk, 2014a;Zhang et al., 2009). Mice that are deficient in decorin or biglycan only have a mild corneal phenotype. ...
The transparent cornea is the major refractive element of the eye. A finely controlled assembly of the stromal extracellular matrix is critical to corneal function, as well as in establishing the appropriate mechanical stability required to maintain corneal shape and curvature. In the stroma, homogeneous, small diameter collagen fibrils, regularly packed with a highly ordered hierarchical organization, are essential for function. This review focuses on corneal stroma assembly and the regulation of collagen fibrillogenesis. Corneal collagen fibrillogenesis involves multiple molecules interacting in sequential steps, as well as interactions between keratocytes and stroma matrix components. The stroma has the highest collagen V:I ratio in the body. Collagen V regulates the nucleation of protofibril assembly, thus controlling the number of fibrils and assembly of smaller diameter fibrils in the stroma. The corneal stroma is also enriched in small leucine-rich proteoglycans (SLRPs) that cooperate in a temporal and spatial manner to regulate linear and lateral collagen fibril growth. In addition, the fibril-associated collagens (FACITs) such as collagen XII and collagen XIV have roles in the regulation of fibril packing and inter-lamellar interactions. A communicating keratocyte network contributes to the overall and long-range regulation of stromal extracellular matrix assembly, by creating micro-domains where the sequential steps in stromal matrix assembly are controlled. Keratocytes control the synthesis of extracellular matrix components, which interact with the keratocytes dynamically to coordinate the regulatory steps into a cohesive process. Mutations or deficiencies in stromal regulatory molecules result in altered interactions and deficiencies in both transparency and refraction, leading to corneal stroma pathobiology such as stromal dystrophies, cornea plana and keratoconus.
Copyright © 2014 Elsevier Ltd. All rights reserved.
... Decorin and biglycan have chondroitin / dermatan sulfate GAG chain attached to their core protein; while lumican, keratocan, mimecan and fibromodulin have keratan sulfate GAG chains. Decorin and keratocan are present in relatively high levels throughout the stroma in mature corneas (Chakravarti et al. 2006;Zhang et al. 2009). In contrast, post-natal keratocytes had low-immunoreactivity for mimecan (Chakravarti et al. 2006). ...
... Decorin and keratocan are present in relatively high levels throughout the stroma in mature corneas (Chakravarti et al. 2006;Zhang et al. 2009). In contrast, post-natal keratocytes had low-immunoreactivity for mimecan (Chakravarti et al. 2006). Fibromodulin has been found in the mouse central cornea at post-natal day (P) 14 but becomes restricted to the limbus by P30 (Chen et al. 2010). ...
... Fibromodulin has been found in the mouse central cornea at post-natal day (P) 14 but becomes restricted to the limbus by P30 (Chen et al. 2010). Similarly, biglycan was expressed highly during corneal development but gradually decreased to low levels in mature cornea (Zhang et al. 2009) and lumican becomes restricted to posterior stroma in adult cornea (Chakravarti et al. 2006). These SLRPs have similar or homologous core protein and along with their GAG chains are also similar in size and thus considered to have a role in maintaining the regular distance between collagen fibrils in the corneal stroma (Hassell and Birk 2010). ...
Several studies have established the role of activated corneal keratocytes in the fibrosis of the cornea. However, the role of keratocytes in maintaining the structural integrity of a normal cornea is less appreciated. We focus on the probable functions of integrins in the eye and of the importance of integrin-mediated keratocyte interactions with stromal matrix in the maintenance of corneal integrity. We point out that further understanding of how keratocytes interact with their matrix could establish a novel direction in preventing corneal pathology including loss of structural integrity as in keratoconus or as in fibrosis of the corneal stroma.
... Class II SLRPs show both temporal and spatial differences. Lumican and keratocan are homogeneous across the corneal stroma at birth, but the expression of lumican is restricted to the posterior stroma after maturation (Chakravarti et al., 2006;Zhang et al., 2009;Chen et al., 2010). Fibromodulin expression in the corneal stroma is strongest at P14, but is decreased and restricted to the peripheral cornea with maturation (Chen et al., 2010). ...
... Genetic mouse models support a role for SLRPs in these human conditions. In addition, the mouse models have demonstrated that SLRPs are critical regulators of collagen fibrillogenesis particularly the linear growth and lateral growth of protofibrils into mature fibrils (Chakravarti et al., 1998;Ezura et al., 2000;Chakravarti et al., 2003Chakravarti et al., , 2006Zhang et al., 2009;Chen et al., 2010;Chen and Birk, 2013). In a decorin-null corneal stroma there is a severe disruption of fibril structure and corneal function while the biglycan-null stroma has a phenotype comparable to wild type stromas (Zhang et al., 2009). ...
... Corneal stromas deficient in class II lumican have a fibril phenotype restricted to the posterior stromal with both fibril structure and transparency disrupted in this region (Chakravarti et al., , 2006. In contrast, deficiency in keratocan or fibromodulin is not associated with a fibril phenotype (Liu et al., 2003;Chen et al., 2010). ...
The corneal stroma is enriched in small leucine-rich proteoglycans (SLRPs), including both class I (decorin and biglycan) and class II (lumican, keratocan and fibromodulin). Transparency is dependent on the assembly and maintenance of a hierarchical stromal organization and SLRPs are critical regulatory molecules. We hypothesize that cooperative interclass SLRP interactions are involved in the regulation of stromal matrix assembly. We test this hypothesis using a compound Bgn(-/o)/Lum(-/-) mouse model and single Lum(-/-) or Bgn(-/o) mouse models and wild type controls. SLRP expression was investigated using immuno-localization and immuno-blots. Structural relationships were defined using ultrastructural and morphometric approaches while transparency was analyzed using in vivo confocal microscopy. The compound Bgn(-/o)/Lum(-/-) corneas demonstrated gross opacity that was not seen in the Bgn(-/o) or wild type corneas and greater than that in the Lum(-/-) mice. The Bgn(-/o)/Lum(-/-) corneas exhibited significantly increased opacity throughout the stroma compared to posterior opacity in the Lum(-/-) and no opacity in Bgn(-/o) or wild type corneas. In the Bgn(-/o)/Lum(-/-) corneas there was abnormal lamellar and fibril structure consistent with the functional deficit in transparency. Lamellar structure was disrupted across the stroma with disorganized fibrils, and altered fibril packing. In addition, fibrils had larger and more heterogeneous diameters with an abnormal structure consistent with abnormal fibril growth. This was not observed in the Bgn(-/o) or wild type corneas and was restricted to the posterior stroma in Lum(-/-) mice. The data demonstrate synergistic interclass regulatory interactions between lumican and biglycan. These interactions are involved in regulating both lamellar structure as well as collagen fibrillogenesis and therefore, corneal transparency.
... Keratocan has a constant temporal and spatial expression pattern in corneal development and the mature cornea [56]. In contrast, during development, lumican is homogenous in both anterior and posterior stroma, but becomes restricted to the posterior stroma in the adult animal [72]. Fibromodulin is not considered a corneal component, however, it has a narrow window of expression extending into the central cornea during early postnatal development [73]. ...
... Fibromodulin is not considered a corneal component, however, it has a narrow window of expression extending into the central cornea during early postnatal development [73]. The class III SLRP, osteoglycin, is only localized to the epithelium and basement membrane zone [72]. The complex expression patterns of SLRPs during corneal development are tightly regulated. ...
... During tendon development, fibromodulin facilitates the growth of protofibrils into mature fibrils [76]. Binding of SLRPs on the collagen fibril surface regulates fibril growth steps and a common feature in SLRP-null models is dysfunctional regulation of fibril diameter and altered fibril structure [2,72,76,92]. ...
Small leucine rich proteoglycans (SLRPs) are involved in a variety of biological and pathological processes. This review focuses on their regulatory roles in matrix assembly. SLRPs have protein cores and hypervariable glycosylation with multivalent binding abilities. During development, differential interactions of SLRPs with other molecules results in tissue-specific spatial and temporal distributions. The changing expression patterns play a critical role in the regulation of tissue-specific matrix assembly and, therefore, tissue function. SLRPs have significant structural roles within extracellular matrices. In addition, they have instructive roles, regulating collagen fibril growth, fibril organization, and extracellular matrix assembly. Moreover, they are involved in mediating cell-matrix interactions. Abnormal SLRP expression and/or structures result in dysfunctional extracellular matrices and pathophysiology. Altered expression of SLRPs has been found in many disease models, and structural deficiency also causes altered matrix assembly. SLRPs regulate the assembly of the extracellular matrix, which defines the microenvironment, modulating both the extracellular matrix and cellular functions leading to an impact on tissue function. © 2013 The Authors Journal compilation © 2013 FEBS.
... Immuno-localization of osteoglycin in the mouse cornea showed localization primarily to the epithelium and epithelial basement membrane with minor stromal localization. In the stroma, osteoglycin was primarily localized to the keratocytes (Chakravarti et al., 2006). Therefore, the stromal role of this SLRP is ambiguous. ...
... SLRPs are regulators of collagen fibril assembly. They have been shown to be critical in regulating linear and lateral growth of protofibrils into mature fibrils in the corneal stroma as well as other tissues (Chakravarti et al., 1998(Chakravarti et al., , 2006Chen and Birk, 2013;Chen et al., 2010;Ezura et al., 2000;Mienaltowski and Birk, 2014;Zhang et al., 2009). Fig. 9 presents a model illustrating SLRP and collagen fibril interactions in the regulation of stromal fibril growth. ...
No other tissue in the body depends more on the composition and organization of the extracellular matrix (ECM) for normal structure and function than the corneal stroma. The precise arrangement and orientation of collagen fibrils, lamellae and keratocytes that occurs during development and is needed in adults to maintain stromal function is dependent on the regulated interaction of multiple ECM components that contribute to attain the unique properties of the cornea: transparency, shape, mechanical strength, and avascularity. This review summarizes the contribution of different ECM components, their structure, regulation and function in modulating the properties of the corneal stroma. Fibril forming collagens (I, III, V), fibril associated collagens with interrupted triple helices (XII and XIV), network forming collagens (IV, VI and VIII) as well as small leucine-rich proteoglycans (SLRP) expressed in the stroma: decorin, biglycan, lumican, keratocan, and fibromodulin are some of the ECM components reviewed in this manuscript. There are spatial and temporal differences in the expression of these ECM components, as well as interactions among them that contribute to stromal function. Unique regions within the stroma like Bowman's layer and Descemet's layer are discussed. To define the complexity of corneal stroma composition and structure as well as the relationship to function is a daunting task. Our knowledge is expanding, and we expect that this review provides a comprehensive overview of current knowledge, definition of gaps and suggests future research directions.
... Lumican deficiency leads to abnormal development and arrangement of collagen fibers in the posterior stroma and leads to decreased corneal transparency. 11,12 We suggest that the decrease in lumican labeling in CCO may reflect its role in the large caliber collagen fiber formation present in Peters anomaly and congenital glaucoma specimens. Furthermore, the notable decrease in lumican labeling in the posterior stroma in Peters anomaly seems to correlate with the larger irregular collagen bundles in the posterior stroma present in Peters anomaly. ...
... A decrease in keratan sulfate labeling has been reported for other congenital corneal disorders such as sclerocornea and in the posterior stroma of lumican deficient mice. 11,12,14 Smooth muscle actin is a marker of myofibroblasts and its presence indicates an active healing process. 15 The increased level of smooth muscle actin in Peters anomalyrelated scars in comparison with acquired scars may indicate that the myofibroblastic changes in the keratocytes in Peters anomaly are similar to what is noted in acquired corneal stromal scarring. ...
Purpose: To examine the immunohistochemical alterations in the
corneal stroma in Peters anomaly (PA) and congenital glaucoma
(CG) compared with age-matched normals and acquired adult
corneal scarring (AACS).
Methods: The clinical features of PA and CG patients who
underwent penetrating keratoplasty were recorded. Immunohistochemistry
of cornea and control tissue (normal and acquired corneal
scars) was performed with antibodies against collagen types I, III,
keratan sulfate, lumican, decorin, and smooth muscle actin followed
by semiquantitative analysis of immunolabeling.
Results: Clinical features in 2 groups were consistent with PA and
CG. Microscopy showed thickened stromal collagen bundles in PA
(n = 15), CG (n = 11), and AACS (n = 20) compared with normals
(n = 18). PA and CG had distinct immunophenotypes compared with
controls. Type I collagen labeling was more intense in CG compared
with PA (intensity grading (IG) 2.73 vs. 2.07; P , 0.001). Decorin,
lumican, and keratan sulfate labeling was significantly less intense in
PA versus AACS (IG; 1.91, 0.38, 1.75 in PA and 2.7, 1.11, 2.61 in
AACS. respectively; P = 0.002, P = 0.001 and P = 0.004) and
normals (IG 1.92, 1.06, 2.59 respectively; P , 0.001, P , 0.001 and
P = 0.005). Collagen I labeling was less intense in CG versus AACS
(IG 2.73 vs. 3.09) (P = 0.007). Collagen III labeling was more
intense in PA/CG than in normals (IG 0.9, 0.64, 0.62 retrospectively)
(P , 0.001 in both).
Conclusions: The immunophenotype of the corneal scar in PA and
CG differs from normal corneas and so does PA from AACS. The
similarities between CG and AACS suggest that CG scarring has an
acquired component.
Key Words: Peters anomaly, congenital glaucoma, immunohistochemistry,
corneal scarring
... Lumican is an important determinant of corneal transparence as shown by the corneal opacity in lumican null mice [241]. Lumican regulates collagen fibril organization, corneal circumferential growth, neutrophil migration in response to bacterial infections, and epithelial cell migration during wound healing and tissue repair [30,131,239,[241][242][243][244][245][246][247]. Mechanistically, lumican protein binds collagen fibrils allowing the highly charged hydrophilic glycosaminoglycans (GAGs) to regulate interfibrillar spacing, an important determinant of corneal transparence [239,242,245]. ...
... Lumican regulates collagen fibril organization, corneal circumferential growth, neutrophil migration in response to bacterial infections, and epithelial cell migration during wound healing and tissue repair [30,131,239,[241][242][243][244][245][246][247]. Mechanistically, lumican protein binds collagen fibrils allowing the highly charged hydrophilic glycosaminoglycans (GAGs) to regulate interfibrillar spacing, an important determinant of corneal transparence [239,242,245]. In spite of its importance, not much is known about the regulation of lumican expression in the cornea. ...
The ocular surface-a continuous epithelial surface with regional specializations including the surface and glandular epithelia of the cornea, conjunctiva, and lacrimal and meibomian glands connected by the overlying tear film-plays a central role in vision. Molecular and cellular events involved in embryonic development, postnatal maturation, and maintenance of the ocular surface are precisely regulated at the level of gene expression by a well-coordinated network of transcription factors. A thorough appreciation of the biological characteristics of the ocular surface in terms of its gene expression profiles and their regulation provides us with a valuable insight into the pathophysiology of various blinding disorders that disrupt the normal development, maturation, and/or maintenance of the ocular surface. This paper summarizes the current status of our knowledge related to the ocular surface development and gene expression and the contribution of different transcription factors to this process.
... The higher magnification sTEM and SEM image of the collagenous constructs (Fig. 4) reveals generally small diameter, polydisperse and laterally fused fibrils (Fig. 4D). Interestingly, the fibrils have an appearance similar to collagen fibrils produced by a proteoglycan (lumican) knock-out mouse [47]. Proteoglycans and their associated glycosaminoglycans have been shown to be directly involved in the control of fibril diameter and spacing. ...
... Proteoglycans and their associated glycosaminoglycans have been shown to be directly involved in the control of fibril diameter and spacing. Therefore, in our system we do not expect to see uniform diameter or circular fibrils without the aid of auxiliary ECM molecules such as proteoglycans, glycosaminoglycans or other types of collagens [47][48][49]. This raises the possibility of using these collagenous matrices to test the effect of knocking-in matrix controlling molecules. ...
... Our study also indicates that this role in structural integrity arises through fibrillar collagen organization, potentially through regulating the thickness of collagen fibres, as demonstrated in the cornea in pioneer studies of lumican. [34][35][36] As lumican can bind to collagen I through leucine-rich repeats 5-7, it is possible that lumican's ability to influence collagen fibre formation could be through direct interaction. 30,31 Other studies also indicate that lumican may have a profibrotic role in cardiac diseases and could induce collagen production, 10,13 yet we have not provided data on this role here. ...
Aims
Familial hypertrophic cardiomyopathy (HCM) is the most common form of inherited cardiac disease. It is characterized by myocardial hypertrophy and diastolic dysfunction, and can lead to severe heart failure, arrhythmias, and sudden cardiac death. Cardiac fibrosis, defined by excessive accumulation of extracellular matrix (ECM) components, is central to the pathophysiology of HCM. The ECM proteoglycan lumican is increased during heart failure and cardiac fibrosis, including HCM, yet its role in HCM remains unknown. We provide an in‐depth assessment of lumican in clinical and experimental HCM.
Methods
Left ventricular (LV) myectomy specimens were collected from patients with hypertrophic obstructive cardiomyopathy (n = 15), and controls from hearts deemed unsuitable for transplantation (n = 8). Hearts were harvested from a mouse model of HCM; Myh6 R403Q mice administered cyclosporine A and wild‐type littermates (n = 8–10). LV tissues were analysed for mRNA and protein expression. Patient myectomy or mouse mid‐ventricular sections were imaged using confocal microscopy, direct stochastic optical reconstruction microscopy (dSTORM), or electron microscopy. Human foetal cardiac fibroblasts (hfCFBs) were treated with recombinant human lumican (n = 3) and examined using confocal microscopy.
Results
Lumican mRNA was increased threefold in HCM patients (P < 0.05) and correlated strongly with expression of collagen I (R² = 0.60, P < 0.01) and III (R² = 0.58, P < 0.01). Lumican protein was increased by 40% in patients with HCM (P < 0.01) and correlated with total (R² = 0.28, P = 0.05) and interstitial (R² = 0.30, P < 0.05) fibrosis. In mice with HCM, lumican mRNA increased fourfold (P < 0.001), and lumican protein increased 20‐fold (P < 0.001) in insoluble ECM lysates. Lumican and fibrillar collagen were located together throughout fibrotic areas in HCM patient tissue, with increased co‐localization measured in patients and mice with HCM (patients: +19%, P < 0.01; mice: +13%, P < 0.01). dSTORM super‐resolution microscopy was utilized to image interstitial ECM which had yet to undergo overt fibrotic remodelling. In these interstitial areas, collagen I deposits located closer to (−15 nm, P < 0.05), overlapped more frequently with (+7.3%, P < 0.05) and to a larger degree with (+5.6%, P < 0.05) lumican in HCM. Collagen fibrils in such deposits were visualized using electron microscopy. The effect of lumican on collagen fibre formation was demonstrated by adding lumican to hfCFB cultures, resulting in thicker (+53.8 nm, P < 0.001), longer (+345.9 nm, P < 0.001), and fewer (−8.9%, P < 0.001) collagen fibres.
Conclusions
The ECM proteoglycan lumican is increased in HCM and co‐localizes with fibrillar collagen throughout areas of fibrosis in HCM. Our data suggest that lumican may promote formation of thicker collagen fibres in HCM.
... Multipotent neural crest cells migrate into the space between the epithelium and the lens vesicle to produce the stromal keratocytes and the single cell layered endothelium (25)(26)(27). The stroma produced by the keratocytes, is composed of glycoproteins, proteoglycans and collagen types I and V primarily that form uniformly thin collagen fibrils organized into orthogonally stacked lamellae to make up a unique ECM that is both transparent and refractive (28,29). In a healthy adult cornea, the stromal keratocytes are quiescent and there is very little turnover of collagens and therefore collagen transcripts are barely detectable, although these are some of the most abundant proteins in the cornea. ...
The cornea is a protective and refractive barrier in the eye crucial for vision. Understanding the human cornea in health, disease and cell-based treatments can be greatly advanced with cornea organoids developed in culture from induced pluripotent stem cells. While a limited number of studies have investigated the single-cell transcriptomic composition of the human cornea, its organoids have not been examined similarly. Here we elucidated the transcriptomic cell fate map of 4-month-old human cornea organoids and human donor corneas. The organoids harbor cell clusters that resemble cells of the corneal epithelium, stroma and endothelium with sub-populations that capture signatures of early developmental states. Unlike the adult cornea where the largest cell population is stromal, the organoids contain large proportions of epithelial and endothelial-like cells. These corneal organoids offer a three-dimensional model to study corneal diseases and integrated responses of different cell types. Significance There is a strong effort to develop cellular surrogates for understanding human corneal diseases. While the three major corneal cell types, epithelial, stromal keratocyte and endothelial cells are often investigated in single monolayer cultures, their interactions in the native tissue impact their differentiation and functions. We developed 3D organoids from human induced pluripotent stem cells to investigate the co-differentiation of corneal cells. Our single cell RNA-sequencing of human donor corneas and the organoids, shows the presence of all three cell types in the organoids and overall, their closer resemblance to developing, rather than adult corneas. These organoids, defined at the single-cell level, offer an experimental system to model human diseases in tissue culture, minimizing the need for valuable animal models.
... Multipotent neural crest cells migrate into the space between the epithelium and the lens vesicle to produce the stromal keratocytes and the single cell layered endothelium (25)(26)(27). The stroma produced by the keratocytes, is composed of glycoproteins, proteoglycans and collagen types I and V primarily that form uniformly thin collagen fibrils organized into orthogonally stacked lamellae to make up a unique ECM that is both transparent and refractive (28,29). In a healthy adult cornea, the stromal keratocytes are quiescent and there is very little turnover of collagens and therefore collagen transcripts are barely detectable, although these are some of the most abundant proteins in the cornea. ...
The cornea is a protective and refractive barrier in the eye crucial for vision. Understanding the human cornea in health, disease and cell-based treatments can be greatly advanced with cornea organoids developed in culture from induced pluripotent stem cells. While a limited number of studies have investigated the single-cell transcriptomic composition of the human cornea, its organoids have not been examined similarly. Here we elucidated the transcriptomic cell fate map of 4-month-old human cornea organoids and human donor corneas. The organoids harbor cell clusters that resemble cells of the corneal epithelium, stroma and endothelium with sub-populations that capture signatures of early developmental states. Unlike the adult cornea where the largest cell population is stromal, the organoids contain large proportions of epithelial and endothelial-like cells. These corneal organoids offer a three-dimensional model to study corneal diseases and integrated responses of different cell types.
... osteoglycin. 9,[13][14][15][16][17][18] The small leucine-rich proteoglycans serve as critical modulators of cell growth and regulate collagen fibrillogenesis, and thereby are important tissue organizers. They also modulate growth factors, including transforming growth factor (TGF)β-1, TGFβ-2, and possibly TGFβ-3. ...
Purpose:
To highlight the cellular, matrix, and hydration changes associated with opacity that occurs in the corneal stroma after injury.
Methods:
Review of the literature.
Results:
The regulated transition of keratocytes to corneal fibroblasts and myofibroblasts, and of bone marrow-derived fibrocytes to myofibroblasts, is in large part modulated by transforming growth factor beta (TGFβ) entry into the stroma after injury to the epithelial basement membrane (EBM) and/or Descemet's membrane. The composition, stoichiometry, and organization of the stromal extracellular matrix components and water is altered by corneal fibroblast and myofibroblast production of large amounts of collagen type I and other extracellular matrix components-resulting in varying levels of stromal opacity, depending on the intensity of the healing response. Regeneration of EBM and/or Descemet's membrane, and stromal cell production of non-EBM collagen type IV, reestablishes control of TGFβ entry and activity, and triggers TGFβ-dependent myofibroblast apoptosis. Eventually, corneal fibroblasts also disappear, and repopulating keratocytes reorganize the disordered extracellular matrix to reestablish transparency.
Conclusions:
Injuries to the cornea produce varying amounts of corneal opacity depending on the magnitude of cellular and molecular responses to injury. The EBM and Descemet's membrane are key regulators of stromal cellularity through their modulation of TGFβ. After injury to the cornea, depending on the severity of the insult, and possibly genetic factors, trace opacity to severe scarring fibrosis develops. Stromal cellularity, and the functions of different cell types, are the major determinants of the level of the stromal opacity.
... Mice lacking both DCN and BGN produce abnormal collagen fibrils, characterized by uncontrolled lateral fibril assembly, which in turn results in fibrils with enormous diameters and aberrant profiles [64,[99][100][101]. BGN can regulate collagen fibrillogenesis in vivo (cornea, tendon) and in vitro, compensating for the loss of DCN [67,102]. ...
Two small leucine-rich proteoglycans (SLRP), decorin and biglycan, play important roles in structural–functional integrity of the placenta and fetal membranes, and their alterations can result in several pregnancy-associated diseases. In this review, we briefly discuss normal placental structure and functions, define and classify SLRPs, and then focus on two SLRPs, decorin (DCN) and biglycan (BGN). We discuss the consequences of deletions/mutations of DCN and BGN. We then summarize DCN and BGN expression in the pregnant uterus, myometrium, decidua, placenta, and fetal membranes. Actions of these SLRPs as ligands are then discussed in the context of multiple binding partners in the extracellular matrix and cell surface (receptors), as well as their alterations in pathological pregnancies, such as preeclampsia, fetal growth restriction, and preterm premature rupture of membranes. Lastly, we raise some unanswered questions as food for thought.
... It has been shown that when CSSCs and hCFs are grown under equivalent culture conditions, secretion of collagen types I, V, and VI, the major protein components of the corneal stromal tissue, was much less abundant in the hCF-secreted constructs than in those produced by hCSSCs. Furthermore, most studies have reported that the ECM secreted by hCFs is lacking the cornea-specific keratan sulfate proteoglycans, keratocan and lumican, that are critical to the regulation of inter-fibril spacing and thus for corneal transparency [214][215][216][217][218][219][220]. ...
Each day, about 2000 U.S. workers have a job-related eye injury requiring medical treatment. Corneal diseases are the fifth cause of blindness worldwide. Most of these diseases can be cured using one form or another of corneal transplantation, which is the most successful transplantation in humans. In 2012, it was estimated that 12.7 million people were waiting for a corneal transplantation worldwide. Unfortunately, only 1 in 70 patients received a corneal graft that same year. In order to provide alternatives to the shortage of graftable corneas, considerable progress has been achieved in the development of living corneal substitutes produced by tissue engineering and designed to mimic their in vivo counterpart in terms of cell phenotype and tissue architecture. Most of these substitutes use synthetic biomaterials combined with immortalized cells, which makes them dissimilar from the native cornea. However, studies have emerged that describe the production of tridimensional (3D) tissue-engineered corneas using untransformed human corneal epithelial cells grown on a totally natural stroma synthesized by living corneal fibroblasts, that also show appropriate histology and expression of both extracellular matrix (ECM) components and integrins. This review highlights contributions from laboratories working on the production of human tissue-engineered corneas (hTECs) as future substitutes for grafting purposes. It overviews alternative models to the grafting of cadaveric corneas where cell organization is provided by the substrate, and then focuses on their 3D counterparts that are closer to the native human corneal architecture because of their tissue development and cell arrangement properties. These completely biological hTECs are therefore very promising as models that may help understand many aspects of the molecular and cellular mechanistic response of the cornea toward different types of diseases or wounds, as well as assist in the development of novel drugs that might be promising for therapeutic purposes.
... Our data show that addition of decorin at a weight ratio of 1:15 with collagen yields thinner fibrils in a looser network, suggesting a potential dose dependence that needs to be further investigated. For lumican, Rada et al. found that its addition into the collagen network in vitro resulted in thinner collagen fibrils, as visualized by transmission electron microscopy 33 , while in vivo studies by Chakravarti et al. showed that the diameter of collagen in corneal stroma was increased in lumicandeficient mice 50 . Our data suggest that lumican has minimal effect on collagen fiber diameter, but there are no other SEM data published for comparison. ...
The extracellular matrix (ECM) is a complex mixture composed of fibrillar collagens as well as additional protein and carbohydrate components. Proteoglycans (PGs) contribute to the heterogeneity of the ECM and play an important role in its structure and function. While the small leucine rich proteoglycans (SLRPs), including decorin and lumican, have been studied extensively as mediators of collagen fibrillogenesis and organization, the function of large matrix PGs in collagen matrices is less well known. In this study, we showed that different matrix PGs have distinct roles in regulating collagen behaviors. We found that versican, a large chondroitin sulfate PG, promotes collagen fibrillogenesis in a turbidity assay and upregulates cell-mediated collagen compaction and reorganization, whereas aggrecan, a structurally-similar large PG, has different and often opposing effects on collagen. Compared to versican, decorin and lumican also have distinct functions in regulating collagen behaviors. The different ways in which matrix PGs interact with collagen have important implications for understanding the role of the ECM in diseases such as fibrosis and cancer, and suggest that matrix PGs are potential therapeutic targets.
... They have abnormally thick collagen fibrils. Lumican maintains the structure of collagen fibrils mainly in the posterior stroma [137]. ...
Corneal stromal wound healing is a complex event that occurs to restore the transparency of an injured cornea. It involves immediate apoptosis of keratocytes followed by their activation, proliferation, migration, and trans-differentiation to myofibroblasts. Myofibroblasts contract to close the wound and secrete extracellular matrix and proteinases to remodel it. Released proteinases may degenerate the basement membrane allowing an influx of cytokines from overlying epithelium. Immune cells infiltrate the wound to clear cellular debris and prevent infections. Gradually basement membrane regenerates, myofibroblasts and immune cells disappear, abnormal matrix is resorbed, and transparency of the cornea is restored. Often this cascade deregulates and corneal opacity results. Factors that prevent corneal opacity after an injury have always intrigued the researchers. They hold clinical relevance as they can guide the outcomes of corneal surgeries. Studies in the past have shed light on the role of various factors in stromal healing. TGFβ (transforming growth factor-beta) signaling is the central player guiding stromal responses. Other major regulators include myofibroblasts, basement membrane, collagen fibrils, small leucine-rich proteoglycans, biophysical cues, proteins derived from extracellular matrix, and membrane channels. The knowledge about their roles helped to develop novel therapies to prevent corneal opacity. This article reviews the role of major regulators that determine the outcome of stromal healing. It also discusses emerging therapies that modulate the role of these regulators to prevent stromal opacity.
... Our data show that addition of decorin at a ratio of 1:15 with collagen yields thinner fibrils in a looser network, suggesting a potential dose dependence that needs to be further investigated. For lumican, Rada et al. found that its addition into the collagen network in vitro resulted in thinner collagen fibrils, as visualized by transmission electron microscopy [33], while in vivo studies by Chakravarti et al. showed that the diameter of collagen in corneal stroma was increased in lumican-deficient mice [49]. Our data suggest that lumican has minimal effect on collagen fiber diameter, but there are no other SEM data published for comparison. ...
The extracellular matrix (ECM) is a complex mixture composed of fibrillar collagens as well as additional protein and carbohydrate components. Proteoglycans (PGs) contribute to the heterogeneity of the ECM and play an important role in its structure and function. While the small leucine rich proteoglycans (SLRPs), including decorin and lumican, have been studied extensively as mediators of collagen fibrillogenesis and organization, the function of large matrix PGs in collagen matrices is less well known. In this study, we showed that different matrix PGs have distinct roles in regulating collagen behaviors. We found that versican, a large chondroitin sulfate PG, promotes collagen fibrillogenesis in a turbidity assay and upregulates cell-mediated collagen compaction and reorganization, whereas aggrecan, a structurally-similar large PG, has different and often opposing effects on collagen. Compared to versican, decorin and lumican also have distinct functions in regulating collagen behaviors. The different ways in which matrix PGs interact with collagen have important implications for understanding the role of the ECM in diseases such as fibrosis and cancer, and suggest that matrix PGs are potential therapeutic targets.
Highlights
Small leucine rich proteoglycans (SLRPs) and large chondroitin sulfate (CS) proteoglycans (PGs) have distinct effects on collagen fibrous network behavior.
Unlike other matrix proteoglycans, versican promotes collagen fibrillogenesis in an in vitro spectrophotometric (turbidity) assay.
The versican core protein has a larger impact on collagen behavior in a fibrillogenesis assay than its glycosaminoglycan chains do.
Versican increases the diameter of collagen fibers and the porosity of collagen fibrous networks, unlike aggrecan and SLRPs.
The addition of versican to collagen does not alter fibroblast contractility but leads to enhanced cell-mediated collagen reorganization and contraction.
... 18 While exact functions are often difficult to discern due to spatial overlap, temporal shifts, and partial compensation between PGs, knockout studies have revealed a broad range of phenotypes. SLRPs alter assembly kinetics, 19,20 tissue hydration, 21,22 and structural mechanics, [23][24][25] while also regulating fibril diameter, 24,26 interfibrillar spacing, 23,27 and enzyme activity of both lysyl oxidase 28,29 and matrix metalloproteinases. 30,31 The multitude of physiological roles attained by PGs are primarily attributable to either steric hindrance or the hygroscopic nature of the GAG chains. ...
Background: Collagenous tissues are composed of precisely oriented, tightly packed collagen fibril bundles to confer the maximal strength within the smallest volume. While this compact form benefits mobility, it consequentially restricts vascularity and cell density to a minimally viable level in some regions. These tissues reside in a homeostatic state with an unstable equilibrium, where perturbations to structure or molecular milieu cause descension into a long-term compromised state. Several studies have shown that glycosaminoglycans are key molecules required for healthy tissue maintenance. Our long-term goal is to determine if glycosaminoglycans serve a critical function of stabilizing soluble monomeric collagen in the interstitial fluid that bathes tissue for immediate availability in tissue development and repair in vivo. Materials and Methods: To test glycosaminoglycan and collagen interactions at the most fundamental level, we have explored the effect of the monosaccharides that populate the glycosaminoglycans of the extracellular matrix on collagen assembly kinetics, pre-established matrix stability, and collagen incorporation into a preassembled matrix. Results: Results showed that monosaccharides increased the threshold concentration required for spontaneous polymerization by at least three orders of magnitude. When the monosaccharides were introduced to a pre-existing collagen network, fibrillar dissociation was undetectable. Fluorescent-labeling studies illustrated that in the presence of the saccharide solution, soluble collagen maintains the functional capacity to integrate into a pre-existing network. Conclusion: This work demonstrates a feasible role for glycosaminoglycans in supporting tissue remodeling and highlights the potential importance of age-related deterioration of glycosaminoglycan biosynthesis in reference to the homeostasis of collagen-based tissues.
... PGs are well known to play a role in maintaining a transparent cornea, with knockout studies showing the fusion of adjacent collagen fibrils and increased opacity of the cornea [35][36][37] . Specifically, lumican has previously been shown to prevent lateral fibril growth of newly deposited collagen fibril intermediates in mouse neonatal development, supporting the role of PGs regulating the diameter of collagen fibrils 38 . Within avian development, the increase in sulphation of GAGs on keratan sulphate PGs is thought to achieve matrix hydration and transparency 39 . ...
As the outer lens in the eye, the cornea needs to be strong and transparent. These properties are governed by the arrangement of the constituent collagen fibrils, but the mechanisms of how this develops in mammals is unknown. Using novel 3-dimensional scanning and conventional transmission electron microscopy, we investigated the developing mouse cornea, focusing on the invading cells, the extracellular matrix and the collagen types deposited at different stages. Unlike the well-studied chick, the mouse cornea had no acellular primary stroma. Collagen fibrils initially deposited at E13 from the presumptive corneal stromal cells, become organised into fibril bundles orthogonally arranged between cells. Extensive cell projections branched to adjacent stromal cells and interacted with the basal lamina and collagen fibrils. Types I, II and V collagen were expressed from E12 posterior to the surface ectoderm, and became widespread from E14. Type IX collagen localised to the corneal epithelium at E14. Type VII collagen, the main constituent of anchoring filaments, was localised posterior to the basal lamina. We conclude that the cells that develop the mouse cornea do not require a primary stroma for cell migration. The cells have an elaborate communication system which we hypothesise helps cells to align collagen fibrils.
... GSEA revealed the Wnt-dependent secretome to be enriched, in both cell lines, for proteins that play key roles in ECM organizational structure and ECM: cell receptor interactions (Supplemental Figure 3). Notably (33,43) and TNC (44)(45)(46)). Thus, these data show that the secretome of TC32 cells is particularly responsive to canonical Wnt activation, and secretion of structural proteins that contribute to ECM organization and remodeling is significantly increased. ...
Tumor: tumor microenvironment (TME) interactions are critical for tumor progression and the composition and structure of the local extracellular matrix (ECM) are key determinants of tumor metastasis. We recently reported that activation of Wnt/beta-catenin signaling in Ewing sarcoma cells induces widespread transcriptional changes that are associated with acquisition of a metastatic tumor phenotype. Significantly, ECM protein-encoding genes were found to be enriched among Wnt/beta-catenin induced transcripts, leading us to hypothesize that activation of canonical Wnt signaling might induce changes in the Ewing sarcoma secretome. To address this hypothesis, conditioned media from Ewing sarcoma cell lines cultured in the presence or absence of Wnt3a was collected for proteomic analysis. Label-free mass spectrometry was used to identify and quantify differentially secreted proteins. We then used in silico databases to identify only proteins annotated as secreted. Comparison of the secretomes of two Ewing sarcoma cell lines revealed numerous shared proteins, as well as a degree of heterogeneity, in both basal and Wnt-stimulated conditions. Gene set enrichment analysis of secreted proteins revealed that Wnt stimulation reproducibly resulted in increased secretion of proteins involved in ECM organization, ECM receptor interactions, and collagen formation. In particular, Wnt-stimulated Ewing sarcoma cells upregulated secretion of structural collagens, as well as matricellular proteins, such as the metastasis-associated protein, tenascin C (TNC). Interrogation of published databases confirmed reproducible correlations between Wnt/beta-catenin activation and TNC and COL1A1 expression in patient tumors. In summary, this first study of the Ewing sarcoma secretome reveals that Wnt/beta-catenin activated tumor cells upregulate secretion of ECM proteins. Such Wnt/beta-catenin mediated changes are likely to impact on tumor: TME interactions that contribute to metastatic progression.
... По изменению состава протеогликанов в роговице накоплено меньше данных, хотя сообщения о сжатии пространства между волокнами коллагена в глубоких слоях стромы по мере старения [23,24] могут служить косвенным признаком участия протеогликанов в этом процессе, как это наблюдается, например, в сухожилии хвоста крысы [25]. Результаты опытов на грызунах (2005,2006) свидетельствуют о том, что изменение паттерна экспрессии протеогликанов, содержащих хондроитинсульфат и кератансульфат, является одним из основных механизмов, регулирующих развитие и формирование архитектоники стромы роговицы в неонатальном периоде [26,27]. ...
The sclera and cornea form the fibrous tunic of the eye which resists external and internal actions and maintains the shape of the eyeball. The functioning of these tunics is associated with their structural and biomechanical properties. Age-related changes of these properties affect protective and supporting functions of the sclera and cornea thus being direct or indirect cause of many ocular disorders.
Structural changes of the fibrous tunic of the eye primarily consist in the disorientation of collagen, elastin,
and proteoglycan fibers which are the basis of corneal and scleral stroma. Fibrils are disoriented, the number
of cross-link bridges increases. Structural changes of ocular connective tissue affect its functionality, i.e., viscoelastic properties of the cornea and sclera.
According to the foreign authors, a common tendency of age-related changes of the fibrous tunic of the eye is
its stiffening and decrease in viscoelastic properties.
... The SLRPs instruct collagen fibril growth, organization, and extracellular matrix assembly (Kalamajski and Oldberg, 2010;Ameye and Young, 2002;Chen and Birk, 2013), binding their leucine-rich repeat regions directly to collagen monomers to generate highly specialized connective tissue matrices (Chakravarti et al, 2006;Chen et al, 2010;Ezura et al, 2000). SLRPs have not only a structural role in regulating the biomaterial properties of collagen-rich ECM, but also a signaling role through direct interaction with growth factors (Iozzo and Schaefer, 2010) and multiple receptors (Santra, Reed, and Iozzo, 2002;Goldoni and Iozzo, 2008;Schonherr et al, 2005;Schaefer et al, 2010). ...
Background:
There are many patients that exhibit connective tissue related cardiac malformations but do not have mutations in collagen genes. The Small Leucine Rich Proteoglycans (SLRP) fibromodulin (FMOD) and lumican (LUM) bind collagen and regulate fibril assembly in other biological contexts.
Results:
FMOD deficient mice and double deficient FMOD;LUM mice exhibited anomalies in regions where cardiac valve tissue interdigitates with adjacent muscle for support. Ectopic connective and/or myocardial tissue(s) was associated with the more severe cardiac valve anomalies in FMOD;LUM deficient mice. At postnatal day 0 (P0) there was an increase in the mesenchymal cell number in the regions where valve cusps anchor in FMOD;LUM deficient mice compared to WT. The cardiac valve anomalies correlated with the highest levels of FMOD expression in the heart and also where myotendinous junctions (MTJ) components biglycan, collagen type I alpha 1, and collagen type VI, are also localized.
Conclusion:
The postnatal assembly of the collagen-rich ECM in regions where cardiac valves anchor, that we have designated 'myotendinous-like junctions' (MTLJ) requires the SLRPs FMOD and LUM. Moreover, FMOD and LUM may facilitate mesenchymal cell differentiation in late stages of cardiac valve development. This article is protected by copyright. All rights reserved.
... Other important proteins that regulate the spatial arrangement and size of collagen fibrils in the cornea stroma are proteoglycans, which bind to specific receptor sites [46,47]. Mice with deficiency in lumican, a type of proteoglycan, showed abnormal fibril structure in the developing cornea [48]. Decorin and dermatan sulfate play a role in the slippage of collagen fibrils over each other [49,50] and in collagen fibrillar structural arrangement [51], respectively. ...
The objective of tissue engineering is to regenerate functional tissues. Engineering functional tissues requires an understanding of the mechanisms that guide the formation and evolution of structure in the extracellular matrix (ECM). In particular, the three-dimensional (3D) collagen fiber arrangement is important as it is the key structural determinant that provides mechanical integrity and biological function. In this review, we survey the current knowledge on collagen organization mechanisms that can be applied to create well-structured functional lamellar tissues and in particular intervertebral disc and cornea. Thus far, the mechanisms behind the formation of cross-aligned collagen fibers in the lamellar structures is not fully understood. We start with cell-induced collagen alignment and strain-stabilization behavior mechanisms which can explain a single anisotropically aligned collagen fiber layer. These mechanisms may explain why there is anisotropy in a single layer in the first place. However, they cannot explain why a consecutive collagen layer is laid down with an alternating alignment. Therefore, we explored another mechanism, called liquid crystal phasing. While dense concentrations of collagen show such behavior, there is little evidence that the conditions for liquid crystal phasing are actually met in vivo. Instead, lysyl aldehyde-derived collagen cross-links have been found essential for correct lamellar matrix deposition. Furthermore, we suggest that supra-cellular (tissue-level) shear stress may be instrumental in the alignment of collagen fibers. Understanding the potential mechanisms behind the lamellar collagen structure in connective tissues will lead to further improvement of the regeneration strategies of functional complex lamellar tissues.
... SLRPs are implicated in regulating assembly of collagen fibrils and the highly organized extracellular matrix essential for corneal transparency (Chakravarti et al., 2006). Lumicans are thought to regulate collagenous matrix assembly in connective tissues via their bi-functional character. ...
... The swelling effect of collagenase treated samples was also reported by Hong et al. [48]. In addition, a proteoglycan-deficient study by Chakravarti et al. [49] reported that lumican-deficient posterior stroma displayed increased fibril diameter and large fibril aggregates. These data could explain why the Ch ase ABC treatment resulted in a larger range of fiber diameters, less defined fibril edges and the partial damage of the D-bandings. ...
Collagen crosslinking is a relatively new treatment for structural disorders of corneal ectasia, such as keratoconus. However, there is a lack of animal models of keratoconus, which has been an obstacle for carefully analyzing the mechanisms of crosslinking and evaluating new therapies. In this study, we treated rabbit eyes with collagenase and chondroitinase enzymes to generate ex vivo corneal ectatic models that simulate the structural disorder of keratoconus. The models were then used to evaluate the protective effect of soluble collagen in the UVA crosslinking system. After enzyme treatment, the eyes were exposed to riboflavin/UVA crosslinking with and without soluble type I collagen. Corneal morphology, collagen ultrastructure, and thermal stability were evaluated before and after crosslinking. Enzyme treatments resulted in corneal curvature changes, collagen ultrastructural damage, decreased swelling resistance and thermal stability, which are similar to what is observed in keratoconus eyes. UVA crosslinking restored swelling resistance and thermal stability, but ultrastructural damage were found in the crosslinked ectatic corneas. Adding soluble collagen during crosslinking provided ultrastructural protection and further enhanced the swelling resistance. Therefore, UVA crosslinking on the ectatic model mimicked typical clinical treatment for keratoconus, suggesting that this model replicates aspects of human keratoconus and could be used for investigating experimental therapies and treatments prior to translation.
... Early in vitro studies of reconstituted collagen fibrils, showed that the presence of lumican and decorin protein cores had an effect on the size of the collagen fibrils: fibrils reconstituted in the presence of the protein cores of the proteoglycans were significantly thinner than the fibrils reconstituted in the absence of the cores (Rada et al., 1993). These findings were confirmed in studies of mutant mice unable to produce the necessary amount of lumican, decorin and biglycan in the cornea (Chakravarti et al., 1998(Chakravarti et al., , 2006Zhang et al., 2008). Three-dimensional electron tomographic studies (Parfitt, 2011), showed that, at least in the case of lumican-deficient mice, the increase in fibrils size can be attributed to a lateral association of normal sized collagen fibrils. ...
The corneal stroma plays several pivotal roles within the eye. Optically, it is the main refracting lens and thus has to combine almost perfect transmission of visible light with precise shape, in order to focus incoming light. Furthermore, mechanically it has to be extremely tough to protect the inner contents of the eye. These functions are governed by its structure at all hierarchical levels. The basic principles of corneal structure and transparency have been known for some time, but in recent years x-ray scattering and other methods have revealed that the details of this structure are far more complex than previously thought and that the intricacy of the arrangement of the collagenous lamellae provides the shape and the mechanical properties of the tissue. At the molecular level, modern technologies and theoretical modelling have started to explain exactly how the collagen fibrils are arranged within the stromal lamellae and how proteoglycans maintain this ultrastructure. In this review we describe the current state of knowledge about the three-dimensional stromal architecture at the microscopic level, and about the control mechanisms at the nanoscopic level that lead to optical transparency.
Copyright © 2015. Published by Elsevier Ltd.
... In this study we examined for the first time, the role of the class II SLRP lumican (LUM) in the developing murine heart. Previous studies using mouse models of LUM deficiency have determined its requirement for normal collagen fiber assembly in the skeletal muscle, [14] tendon [15] and cornea [16,17]. We evaluated the spatiotemporal localization of LUM throughout cardiac development and using a LUM deficient mouse model determine a potentially critical role for LUM in murine cardiac development. ...
The ability of the heart to adapt to increased stress is dependent on modification of its extracellular matrix (ECM) architecture that is established during postnatal development as cardiomyocytes differentiate, a process that is poorly understood. We hypothesized that the small leucine-rich proteoglycan (SLRP) lumican (LUM), which binds collagen and facilitates collagen assembly in other tissues, may play a critical role in establishing the postnatal murine myocardial ECM. Although previous studies suggest LUM deficient mice (lum(-/-)) exhibit skin anomalies consistent with Ehlers-Danlos syndrome, lum(-/-) hearts have not been evaluated. These studies show LUM was immunolocalized to non-cardiomyocytes of the cardiac ventricles and its expression increased throughout development. Lumican deficiency resulted in significant (50%) perinatal death and further examination of the lum(-/-) neonatal hearts revealed an increase in myocardial tissue without a significant increase in cell proliferation. However cardiomyocytes from surviving postnatal day 0 (P0), 1 month (1 mo) and adult (4 mo) lum(-/-) hearts were significantly larger than their wild type (WT) littermates. Immunohistochemistry revealed that the increased cardiomyocyte size in the lum(-/-) hearts correlated with alteration of the cardiomyocyte pericellular ECM components collagenα1(I) and the class I SLRP decorin (DCN). Western blot analysis demonstrated that the ratio of glycosaminoglycan (GAG) decorated DCN to core DCN was reduced in P0 and 1 mo lum(-/-) hearts. There was also a reduction in the β and γ forms of collagenα1(I) in lum(-/-) hearts. While the total insoluble collagen content was significantly reduced, the fibril size was increased in lum(-/-) hearts, indicating LUM may play a role in collagen fiber stability and lateral fibril assembly. These results suggest that LUM controls cardiomyocyte growth by regulating the pericellular ECM and also indicates that LUM may coordinate multiple factors of collagen assembly in the murine heart. Further investigation into the role of LUM may yield novel therapeutic targets and/or biomarkers for patients with cardiovascular disease.
Copyright © 2015. Published by Elsevier Ltd.
... This expression of keratocan and keratan sulfate is of particular significance. The uniform spacing of collagen fibrils in the corneal stromal extracellular matrix is regulated by proteoglycans [38][39][40][41][42][43]. Keratan sulfate (KS), a sulfated glycosaminoglycan constituting 60% of the corneal stromal proteoglycans, is more abundant by at least an order of magnitude in the cornea than in other tissues of the body [23]. ...
Corneal blindness afflicts millions of individuals worldwide and is currently treated by grafting with cadaveric tissues; however, there are worldwide donor tissue shortages, and many allogeneic grafts are eventually rejected. Autologous stem cells present a prospect for personalized regenerative medicine and an alternative to cadaveric tissue grafts. Dental pulp contains a population of adult stem cells and, similar to corneal stroma, develops embryonically from the cranial neural crest. We report that adult dental pulp cells (DPCs) isolated from third molars have the capability to differentiate into keratocytes, cells of the corneal stoma. After inducing differentiation in vitro, DPCs expressed molecules characteristic of keratocytes, keratocan, and keratan sulfate proteoglycans at both the gene and the protein levels. DPCs cultured on aligned nanofiber substrates generated tissue-engineered, corneal stromal-like constructs, recapitulating the tightly packed, aligned, parallel fibrillar collagen of native stromal tissue. After injection in vivo into mouse corneal stroma, human DPCs produced corneal stromal extracellular matrix containing human type I collagen and keratocan and did not affect corneal transparency or induce immunological rejection. These findings demonstrate a potential for the clinical application of DPCs in cellular or tissue engineering therapies for corneal stromal blindness.
©AlphaMed Press.
... Indeed, Lum -/mice develop bilateral corneal opacities together with skin laxity and fragility reminiscent of Ehlers Danlos syndrome [498]. The posterior corneal stroma is most vulnerable to lumican deficiency as this region shows early developmental defects in fibril structure and architecture in the Lum -/mice [499]. The causative role of lumican in corneal opacity is demonstrated by genetic studies where a mouse overexpressing lumican in the cornea, driven by the keratocan promoter, can fully rescue the Lum -/eye phenotype [500]. ...
We provide a comprehensive classification of the proteoglycan gene families and respective protein cores. This updated nomenclature is based on three criteria: Cellular and subcellular location, overall gene/protein homology, and the utilization of specific protein modules within their respective protein cores. These three signatures were utilized to design four major classes of proteoglycans with distinct forms and functions: the intracellular, cell-surface, pericellular and extracellular proteoglycans. The proposed nomenclature encompasses forty-three distinct proteoglycan-encoding genes and many alternatively-spliced variants. The biological functions of these four proteoglycan families are critically assessed in development, cancer and angiogenesis, and in various acquired and genetic diseases where their expression is aberrant.
Copyright © 2015. Published by Elsevier B.V.
... In comparison with sclera, documented research on the fate of human corneal PGs with aging is relatively lacking, although the reported compaction of collagen interfibrillar spacing with age in the deep stroma [84,102] may be indicative of PG involvement, as found in developing rat tail tendon [133]. Extensive work using rodent models has strongly implicated changing expression patterns of both CS and keratan sulfate (KS) PG expression as important regulatory processes in neonatal development and maturation of corneal stromal architecture [22,88]. ...
With increasing age, the mechanical performance of the cornea and sclera is impaired due to structural changes in the major structural proteins, namely collagens , proteoglycans and elastin. In addition, the level of hydration in the ocular tunic decreases over time. These structural changes profoundly impact on the biomechanical properties of the corneo-scleral tunic. This chapter focuses on the structural and biomechanical changes that occur in the corneo-scleral tunic with age. The techniques that are utilized in order to determine the mechanical properties of both the cornea and sclera are discussed, and a comprehensive review of studies which have characterized age-related changes in ocular biomechanics are presented. The cornea is found to increase in stiffness with age and all the characteristics of viscoelastic behavior (creep , stress-relaxation and hysteresis) decrease with age. Similarly, the stiffness of the sclera increases markedly with age although the reported magnitude of stiffening varies significantly from one study to another. This may be related to variations amongst the different techniques that have been utilized. Increased stiffening in the cornea and the sclera with age is strongly associated with the increase in collagen crosslinking that occurs as part of the natural aging process.
... Keratocandeficient mice demonstrate decreased corneal stromal thickness and less organized arrangement of stromal collagen fibers, although normal corneal clarity is maintained [29]. Heterozygous lumican-deficient mice also have clear corneas, but posterior corneal stromal opacification is observed in homozygous lumicandeficient mice and is associated with irregular arrangement of thickened collagen fibrils [30][31][32]. As the corneas of lumicandeficient mice demonstrate decreased levels of keratocan expression, and as the expression of lumican and keratocan are directly related, lumican appears to regulate the transcription of KERA at the promoter level [33]. ...
Posterior amorphous corneal dystrophy (PACD) is a rare, autosomal dominant disorder affecting the cornea and iris. Next-generation sequencing of the previously identified PACD linkage interval on chromosome 12q21.33 failed to yield a pathogenic mutation. However, array-based copy number analysis and qPCR were used to detect a hemizygous deletion in the PACD linkage interval containing 4 genes encoding small leucine-rich proteoglycans (SLRPs): KERA, LUM, DCN, and EPYC. Two other unrelated families with PACD also demonstrated deletion of these SLRPs, which play important roles in collagen fibrillogenesis and matrix assembly. Given that these genes are essential to the maintenance of corneal clarity and the observation that knockout murine models display corneal phenotypic similarities to PACD, we provide convincing evidence that PACD is associated with haploinsufficiency of these SLRPs.
... As shown in Fig. 4, collagen types I, V, and VI, the major protein components stromal tissue, were much less abundant in the hCFsecreted constructs than in those produced by hCSSCs. Furthermore, ECM secreted by hCFs was lacking in cornea-specific keratan sulfate proteoglycans, keratocan and lumican, which are critical to regulate inter-fibril spacing (Funderburgh et al. 1986;Chakravarti et al. 1998;Funderburgh 2000;Kao and Liu 2002;Carlson et al. 2005;Chakravarti et al. 2006). In contrast, hCSSC-secreted ECM was abundant in collagen types I, V and VI as well as keratan sulfate proteoglycans, keratocan and lumican. ...
Recapitulation of human corneal stromal tissue is believed to be among the most challenging steps in engineering human corneal tissue because of the difficulty in reproducing its highly-ordered hierarchical ultrastructure, which imparts its robust biomechanical properties and optical transparency. In this study, we compared the feasibility of utilizing human corneal stromal stem cells (hCSSCs) and human corneal fibroblasts (hCFs) in the generation of human corneal stromal tissue on a highly-aligned fibrous substrate made from poly(ester urethane) urea. In the serum-free keratocyte differentiation medium supplemented with FGF-2 (10 ng/mL) and TGF-β3 (0.1 ng/mL), hCSSCs successfully differentiated into keratocytes and secreted multilayered lamellae with orthogonally-oriented collagen fibrils, in a pattern mimicking human corneal stromal tissue. The constructs were 60∼70 μm thick and abundant in cornea-specific extracellular matrix (ECM) components, including keratan sulfate, lumican, and keratocan. Under the identical conditions, hCFs tended to differentiate into myofibroblasts and deposited a less-organized collagen-fibrillar construct in a pattern with similarities to corneal scar tissue due to a lack of cornea-specific ECM components. These observations demonstrated that hCSSCs showed a much greater potential, under proper substrate and growth factor guidance, to facilitate the generation of a biological human cornea equivalent. Unlike hCSSCs, hCFs were less responsive to these environmental cues and under identical culture conditions generated an ECM that poorly mimicked the native, functional tissue structure and composition.
... Thereby they control TGF-ß induced fibrosis, tumor growth and cell proliferation [184]. Lumican and fibromodulin are critical regulators of collagen fibril organization; lack of lumican in the corneal stroma causes turbation due to collapse of the regular collagen fibril arrangement [185,186]. Fibromodulin is highly negatively charged due to two keratansulfate chains and tyrosine sulphate residues and is therefore able to bind a number of growth factors like FGF-2 and cytokines [161]. ...
Small leucine rich proteoglycans (SLRPs) are the largest family of proteoglycans, with 18 members that are subdivided into five classes. SLRPs are small in size and can be present in tissues as glycosylated and non-glycosylated proteins, and the most studied SLRPs include decorin, biglycan, lumican, keratocan and fibromodulin. SLRPs specifically bind to collagen fibrils, regulating collagen fibrillogenesis and the biomechanical properties of tissues, and are expressed at particularly high levels in fibrous tissues, such as the cornea. However, SLRPs are also very active components of the ECM, interacting with numerous growth factors, cytokines and cell surface receptors. Therefore, SLRPs regulate major cellular processes and have a central role in major fundamental biological processes, such as maintaining corneal homeostasis and transparency and regulating corneal wound healing. Over the years, mutations and/or altered expression of SLRPs have been associated with various corneal diseases, such as congenital stromal corneal dystrophy and cornea plana. Recently, there has been great interest in harnessing the various functions of SLRPs for therapeutic purposes. In this comprehensive review, we describe the structural features and the related functions of SLRPs, and how these affect the therapeutic potential of SLRPs, with special emphasis on the use of SLRPs for treating ocular surface pathologies.
Metastasis remains the primary cause of death in cancer patients. The invasion-metastasis cascade is dependent on crosstalk between tumor cells and the tumor microenvironment, including stromal cells, extracellular matrix, and blood vessels, at both primary and distal sites. Ewing sarcoma is a bone and soft tissue tumor primarily affecting children and adolescents. Patients with only local disease have high survival rates of >70%. However, current treatment strategies for patients developing metastasis have dismal survival rates of < 20%, making it imperative to better understand the biological processes that drive Ewing sarcoma metastasis. Wnt/beta-catenin activation is correlated with worse overall survival in Ewing sarcoma patients and tumor cell autonomous changes in cytoskeletal organization; however, the tumor cell non-autonomous changes induced by Wnt/beta-catenin remain unexplored. Extracellular matrix encoding genes and genes involved in tumor: tumor microenvironment crosstalk are among the genes activated by Wnt/beta-catenin signaling. Therefore, we hypothesized that Wnt/beta-catenin induces changes in the tumor microenvironment to promote Ewing sarcoma progression. The results of our studies have shown that activation of the Wnt/beta-catenin pathway led to increased secretion of structural collagens and matricellular proteins. Intriguingly, in the secretome of Wnt-activated Ewing sarcoma cells we also detected TGF-beta ligands and proteins that are downstream targets of the TGF-beta signaling cascade. We show that activation of Wnt leads to derepression of TGFBR2, the key mediator of TGF-beta signaling, and detect discrete tumor cell sub-populations, responsive to both Wnt and TGF-beta ligands. Studies of Ewing sarcoma models, in vitro and in vivo, as well as in two independent patient cohorts, confirmed a direct relationship between beta-catenin signaling, TGF-beta activation, and angiogenesis in tumor cells. Mechanistically, this is due, in part, to increased endothelial cell proliferation mediated by increased secretion of the matricellular protein tenascin C. Additionally, we evaluated the other potential functions of the matricellular protein, tenascin C, in Ewing sarcoma progression, and identified stress-mediated activation of tenascin C through activation of Src kinase enhances an invasive phenotype. In conclusion, we have elucidated the importance of Wnt/beta-catenin signaling in altering tumor: tumor microenvironment interactions to promote Ewing sarcoma progression, through secretion of proteins such as tenascin C into the tumor microenvironment. These studies highlight tenascin C as a potential “achilles heel” and therapeutic target in Ewing sarcoma progression. Together these studies illustrate the critical contribution of tumor cell heterogeneity, cell plasticity, and tumor: tumor microenvironment crosstalk to sarcoma progression.
Purpose: To present the clinical and cytogenetic features of a previously unreported family with posterior amorphous corneal dystrophy (PACD) associated with a heterozygous deletion of the small leucine-rich proteoglycan (SRLP) genes on chromosome 12.
Methods: Clinical characterization was performed using slit lamp biomicroscopic and optical coherence tomography (OCT) imaging. Genomic DNA was collected from affected and unaffected family members, and a cytogenomic array was used to identify copy number variations (CNV) present in the PACD locus.
Results: Three members of a Guatemalan family presented with clinical characteristics consistent with PACD: bilateral posterior stromal lamellar opacification, decreased corneal curvature, and iridocorneal adhesions. OCT imaging demonstrated decreased corneal thickness and hyperreflectivity of the posterior third of the corneal stroma. CNV analysis confirmed the presumed clinical diagnosis of PACD by revealing a 0.304 Mb heterozygous deletion in the PACD locus on chromosome 12 that included the four SLRP genes (KERA, LUM, DCN, and EPYC) deleted in each of the PACD families in which CNV analysis has been reported.
Conclusions: This is the first report of the OCT appearance of PACD and the second confirmation of a heterozygous deletion of chromosome 12q21.33 as the cause of PACD, highlighting the utility of array-based cytogenomics to confirm the suspected clinical diagnosis of PACD. As the smallest previously reported pathogenic deletion was 0.701 Mb, the 0.304-Mb deletion we report is the smallest identified to date and reduces the size of the PACD locus to 0.275 Mb.
The SLRP opticin (OPTC) has been demonstrated to be produced and degraded in osteoarthritic (OA) human cartilage. Here, we investigated the in vivo effect of OPTC deficiency in OA cartilage. OA was induced in 10-week-old Optc -/- and Optc +/+ mice. Ten weeks post-surgery, cartilage was processed for histology and immunohistochemistry. SLRP expression was determined in non-operated mouse cartilage. OA Optc -/- demonstrated significant protection against cartilage degradation. Data revealed that in non-operated Optc -/- cartilage, expression of SLRPs lumican and epiphycan was up-regulated at day 3 and in 10-week-olds (p ≤ 0.039), and fibromodulin down-regulated in 10-week-olds (p = 0.001). Immunohistochemistry of OA mice showed a similar pattern. In OA Optc -/- cartilage, markers of degradation and complement factors were all down-regulated (p ≤ 0.038). In OA Optc -/- cartilage, collagen fibers were thinner and better organized (p = 0.038) than in OA Optc +/+ cartilage. The protective effect of OPTC deficiency during OA results from an overexpression of lumican and epiphycan, known to bind and protect collagen fibers, and a decrease in fibromodulin, contributing to a reduction in the complement activation/inflammatory process. This work suggests that the evaluation of the composition of the different SLRPs in OA cartilage could be applied as a new tool for OA prognosis classification.
The small leucine-rich proteoglycans (SLRPs), decorin and biglycan, are key regulators of collagen fibril and matrix assembly. The goal of this work was to elucidate the roles of decorin and biglycan in tendon homeostasis. Our central hypothesis is that decorin and biglycan expression in the mature tendon would be critical for the maintenance of the structural and mechanical properties of healthy tendons. Defining the function(s) of these SLRPs in tendon homeostasis requires that effects in the mature tendon be isolated from their influence on development. Thus, we generated an inducible knockout mouse model that permits genetic ablation of decorin and biglycan expression in the mature tendon, while maintaining normal expression during development. Decorin and biglycan expression were knocked out in the mature patellar tendon with the subsequent turnover of endogenous SLRPs deposited prior to induction. The acute absence of SLRP expression was associated with changes in fibril structure with a general shift to larger diameter fibrils in the compound knockout tendons, together with fibril diameter heterogeneity. In addition, tendon mechanical properties were altered. Compared to wild-type controls, acute ablation of both genes resulted in failure of the tendon at lower loads, decreased stiffness, a trend towards decreased dynamic modulus, as well as a significant increase in percent relaxation and tissue viscosity. Collagen fiber realignment was also increased with a delayed and slower in response to load in the absence of expression. These structural and functional changes in response to an acute loss of decorin and biglycan expression in the mature tendon demonstrate a significant role for these SLRPs in adult tendon homeostasis.
Purpose: to evaluate the influence of pseudoexfoliation syndrome (PEX) on the development and clinical course of «dry eye» syndrome.
Material and Methods: This cross-sectional «case-control» study included 240 eyes of 120 patients with PEX and 240 eyes of 120 normal age-matched individuals. Pseudoexfoliation was established by slit-lamp examination with a maximally dilated pupil. To study the tear function Schirmer’s I test and tear film break-up time (TBUT) were performed.
Results. Both Schirmer test and break-up time values were significantly lower in the PEX patients than in the control group. In the PEX-group the mean TBUT result was 7,93±2,14 s, in control group - 10,37±3,0 s (p<0,001). The mean Schirmer’s I test results were 9,55±3,12 mm and 11,63±3,53 mm, respectively (p<0,001). Lid parallel conjunctival folds (LIPCOF) were presented in73,8% of PEX eyes and 52,9% of normal eyes (p=0,000002, І=22,428). TBUT and Schirmer’s I test score showed an inverse correlation with age in both groups. The difference between PEX and individuals was significant in every age group (p<0,001). Significantly lower tear production and TBUT were associated with increasing degree of PEX. Patients with clinically «unilateral» PEX had a lower TBUT in the PEX-positive eye compared with the non-involved fellow eye (8,5±2,1 s and 9,9±1,7 s, relatively) (p=0,001).
Conclusion. Compared with age-matched individuals, PEX was found to be associated with lower scores in Schirmer I and break-up time tests and can be considered as «risk factor» for dry eye syndrome. The degree of TBUT and Schirmer’s changes depends on the stage of PEX.
Structural biomechanical properties of the ocular corneoscleral shell largely determine its anatomic and optical parameters and its supporting and protective function. Therefore, changes related to age restructuring processes may affect the state of the cornea and the sclera, which should be taken into account when diagnosing eye diseases, especially those emerging in old age. As shown by recent literary data, age-related changes of the corneoscleral shell affecting its biomechanical properties involve all connective tissue components of the extracellular matrix: fibrous proteins (collagen and elastin) and intermediate substance components (proteoglycans and glycosaminoglycans). Aged patients have been found to have a larger diameter of elastic fiber fibrils in the external part of the sclera and a lower density of fibrils in the center as compared to young patients, which is an evidence of elastin damage at the molecular level and fibril degeneration. Age-related changes of proteoglycans are primarily manifested in hydration loss, which leads to an increase in corneal and sclera density and regional thinning of tissues. Age-related changes of collagen are less expressed than those of elastin and proteoglycans. Yet, the distance between collagen fibrils in the cornea becomes smaller with age; they are subject to destruction, and small spaces devoid of collagen tend to appear in the posterior stroma. The most pronounced age-related degenerative changes of collagen in the deeper layers of the corneal stroma occur in the limb, which accumulates more cross striated collagen fibrils. Recent years of research have shown that the formation of cross-linked chemical bonds, i.e. intra- and intermolecular cross links of collagen is the most important structural factor. It is this particular process that is responsible for structural stability of the corneal and scleral tissue, which tends to change with age or due to certain eye diseases, such as keratoconus or progressive myopia. Obviously, we need clinical technologies that allow an adequate estimation of the biomechanical state of the fibrous shell of the eye.
Corneal disease is the main cause of blindness and keratoplasty is the only widely accepted treatment. Shortage of donor tissue makes the biomaterials for corneal regeneration a hot area of research. Collagen is the main component of corneal stroma, so collagen becomes a promising material for corneal repair. However, due to the drawbacks of collagen, it needs to be further modified to satisfy the requirement of corneal regeneration. In this article, we highlight the importance of collagen materials for corneal repair, and summarize several methods of preparing collagen based corneal regeneration materials, including chemical crosslinking, plastic compression of collagen, and collagen vitrigel. These modification methods can make collagen membranes with remarkable properties such as enough mechanical and suture retention strength, antibacterial property and excellent optical property. These materials may provide potential treatment for corneal disease. © 2016 Science China Press and Springer-Verlag Berlin Heidelberg
Corneal dystrophies are a group of inherited disorders affecting the cornea, many of which lead to visual impairment. The International Committee for Classification of Corneal Dystrophies has established criteria to clarify the status of the various corneal dystrophies, which include the knowledge of the underlying genetics. In this review, we discuss the International Committee for Classification of Corneal Dystrophies category 1 (second edition) corneal dystrophies, for which a clear genetic link has been established. We highlight the various mechanisms underlying corneal dystrophy pathology, including structural disorganization, instability or maladhesion, aberrant protein stability and deposition, abnormal cellular proliferation or apoptosis, and dysfunction of normal enzymatic processes. Understanding these genetic mechanisms is essential for designing targets for therapeutic intervention, especially in the age of gene therapy and gene editing.
With increasing age, the mechanical performance of the cornea
and sclera
is impaired due to structural changes in the major structural proteins, namely collagens
, proteoglycans
and elastin. In addition, the level of hydration in the ocular
tunic decreases over time. These structural changes profoundly impact on the biomechanical properties of the corneo-scleral tunic. This chapter focuses on the structural and biomechanical changes that occur in the corneo-scleral tunic with age. The techniques that are utilized in order to determine the mechanical properties of both the cornea and sclera are discussed, and a comprehensive review of studies which have characterized age-related changes in ocular biomechanics are presented. The cornea is found to increase in stiffness
with age and all the characteristics of viscoelastic behavior (creep
, stress-relaxation and hysteresis) decrease with age. Similarly, the stiffness of the sclera increases markedly with age although the reported magnitude of stiffening varies significantly from one study to another. This may be related to variations amongst the different techniques that have been utilized. Increased stiffening in the cornea and the sclera with age is strongly associated with the increase in collagen crosslinking that occurs as part of the natural aging process.
Purpose:
To examine the immunohistochemical alterations in the corneal stroma in Peters anomaly (PA) and congenital glaucoma (CG) compared with age-matched normals and acquired adult corneal scarring (AACS).
Methods:
The clinical features of PA and CG patients who underwent penetrating keratoplasty were recorded. Immunohistochemistry of cornea and control tissue (normal and acquired corneal scars) was performed with antibodies against collagen types I, III, keratan sulfate, lumican, decorin, and smooth muscle actin followed by semiquantitative analysis of immunolabeling.
Results:
Clinical features in 2 groups were consistent with PA and CG. Microscopy showed thickened stromal collagen bundles in PA (n = 15), CG (n = 11), and AACS (n = 20) compared with normals (n = 18). PA and CG had distinct immunophenotypes compared with controls. Type I collagen labeling was more intense in CG compared with PA (intensity grading (IG) 2.73 vs. 2.07; P < 0.001). Decorin, lumican, and keratan sulfate labeling was significantly less intense in PA versus AACS (IG; 1.91, 0.38, 1.75 in PA and 2.7, 1.11, 2.61 in AACS. respectively; P = 0.002, P = 0.001 and P = 0.004) and normals (IG 1.92, 1.06, 2.59 respectively; P < 0.001, P < 0.001 and P = 0.005). Collagen I labeling was less intense in CG versus AACS (IG 2.73 vs. 3.09) (P = 0.007). Collagen III labeling was more intense in PA/CG than in normals (IG 0.9, 0.64, 0.62 retrospectively) (P < 0.001 in both).
Conclusions:
The immunophenotype of the corneal scar in PA and CG differs from normal corneas and so does PA from AACS. The similarities between CG and AACS suggest that CG scarring has an acquired component.
Transforming growth factor-β (TGF-β) plays a key role during corneal stroma scarring. It is a critical regulator in abnormal deposition of extracellular matrix (ECM) and construction of collagen fibers. TGF-β is a family cytokines secreted by a variety of cells. It has multiple effects only when it is activated. The major signaling pathway of TGF-β is Smad, which is activated by the binding of TGF-β and its receptors. TGF-β stimulates the synthesis and secretion of collagen and proteoglycan, which results in disbalance of synthesis and degradation of ECM. The mechanism of TGF-β on promoting corneal fibrosis includes upregulating connective tissue growth factor (CTGF), downregulating keratin sulfate proteoglycans(KSPGs) and inhibiting the degradation of ECM by regulating matrix metalloproteinases(MMPs). Recently, TGF-β has been a promoting growth factor of scar-free wound healing because of its extensive biological effects. This review summarized biological characteristics of TGF-β, its receptors and Smads signal pathway and the molecular mechanism that accelerates corneal stroma scarring.
This paper mainly reviews sources discussed in recent years that are devoted to the genetics of corneal development. Genetically caused separation processes of cornea bud and its specification and differentiation are considered. It is shown that mutation disorders in the genes responsible for differentiation of the cornea can lead to different forms of dystrophies and other corneal disorders.
Decorin, a small leucine-rich proteoglycan (SLRP), is involved in the pathophysiology of human congenital stromal corneal dystrophy (CSCD). This disease is characterized by corneal opacities and vision impairment. In reported cases, the human gene encoding decorin contains point mutations in exon 10, generating a truncated form of decorin lacking the C-terminal 33 amino acid residues. We have previously described a transgenic mouse model carrying a similar mutation in the decorin gene that leads to an ocular phenotype characterized by corneal opacities identical to CSCD in humans. We have also identified abnormal synthesis and secretion of various SLRPs in mutant mouse corneas. In the present study, we found that mutant C-terminal truncated decorin was retained in the cytoplasm of mouse keratocytes in vivo and of transfected human embryonic kidney cells. This resulted in endoplasmic reticulum stress and an unfolded protein response. Thus, we propose a novel cell-based mechanism underlying CSCD in which a truncated SLRP protein core is retained intracellularly, its accumulation triggering endoplasmic reticulum stress that results in abnormal SLRP synthesis and secretion, which ultimately affects stromal structure and corneal transparency.
The class of small leucine-rich proteoglycans (SLRPs) is a family of homologous proteoglycans harboring relatively small (36–42
kDa) protein cores compared with the larger cartilage and mesenchymal proteoglycans. SLRPs have been localized to most skeletal
regions, with specific roles designated during all phases of bone formation, including periods relating to cell proliferation,
organic matrix deposition, remodeling, and mineral deposition. This is mediated by key signaling pathways regulating the osteogenic
program, including the activities of TGF-β, bone morphogenetic protein, Wnt, and NF-κB, which influence both the number of
available osteogenic precursors and their subsequent development, differentiation, and function. On the other hand, SLRP depletion
is correlated with degenerative diseases such as osteoporosis and ectopic bone formation. This minireview will focus on the
SLRP roles in bone physiology and pathology.
The distribution, supramolecular form, and arrangement of collagen types I and V in the chicken embryo corneal stroma were studied using electron microscopy, collagen type-specific monoclonal antibodies, and a preembedding immunogold method. Double-label immunoelectron microscopy with colloidal gold-tagged monoclonal antibodies was used to simultaneously localize collagen type I and type V within the chick corneal stroma. The results definitively demonstrate, for the first time, that both collagens are codistributed within the same fibril. Type I collagen was localized to striated fibrils throughout the corneal stroma homogeneously. Type V collagen could be localized only after pretreatment of the tissue to partially disrupt collagen fibril structure. After such pretreatments the type V collagen was found in regions where fibrils were partially dissociated and not in regions where fibril structure was intact. When pretreated tissues were double labeled with antibodies against types I and V collagen coupled to different size gold particles, the two collagens colocalized in areas where fibril structure was partially disrupted. Antibodies against type IV collagen were used as a control and were nonreactive with fibrils. These results indicate that collagen types I and V are assembled together within single fibrils in the corneal stroma such that the interaction of these collagen types within heterotypic fibrils masks the epitopes on the type V collagen molecule. One consequence of the formation of such heterotypic fibrils may be the regulation of corneal fibril diameter, a condition essential for corneal transparency.
The proteoglycans extracted from adult chicken were initially purified by DEAE-chromatography. Digestion of these proteoglycans with chondroitinase ABC generated a single 40-kDa core protein while digestion with keratanase generated a single 52-kDa core protein. Digestion with both enzymes combined, however, increased the amount of 40-kDa core protein produced. This suggested that the 40-kDa core protein exists with chondroitin/dermatan sulfate (C/DS) side chains alone and with both C/DS and keratan sulfate (KS) side chains. The proteoglycan fraction was initially digested with chondroitinase ABC, and the M(r) = 40,000 core protein derived from proteoglycans containing C/DS side chains alone was isolated. Amino-terminal sequencing showed it to be the chick cognate of decorin. The remaining proteoglycans were then digested with keratanase, and both the 40-kDa core protein and the 52-kDa core proteins derived from KS-containing proteoglycans were purified. The M(r) = 40,000 core protein derived from proteoglycans containing both C/DS and KS side chains had the same amino-terminal sequence as decorin and cross-reacted with antibodies to decorin. Sequence from the 52-kDa core protein derived from KS-containing proteoglycans showed it to be lumican. The results of this study suggest that adult chick corneas contain two isoforms of decorin: one containing C/DS side chains and the other, a hybrid, containing both C/DS and KS side chains. Embryonic corneas did not contain the hybrid isoform of decorin. These results suggest that different post-translational modifications occur to the decorin gene product during corneal development and maturation.
The distribution, supramolecular form, and arrangement of collagen types I and V in the chicken embryo corneal stroma were studied using electron microscopy, collagen type-specific monoclonal antibodies, and a preembedding immunogold method. Double-label immunoelectron microscopy with colloidal gold-tagged monoclonal antibodies was used to simultaneously localize collagen type I and type V within the chick corneal stroma. The results definitively demonstrate, for the first time, that both collagens are codistributed within the same fibril. Type I collagen was localized to striated fibrils throughout the corneal stroma homogeneously. Type V collagen could be localized only after pretreatment of the tissue to partially disrupt collagen fibril structure. After such pretreatments the type V collagen was found in regions where fibrils were partially dissociated and not in regions where fibril structure was intact. When pretreated tissues were double labeled with antibodies against types I and V collagen coupled to different size gold particles, the two collagens colocalized in areas where fibril structure was partially disrupted. Antibodies against type IV collagen were used as a control and were nonreactive with fibrils. These results indicate that collagen types I and V are assembled together within single fibrils in the corneal stroma such that the interaction of these collagen types within heterotypic fibrils masks the epitopes on the type V collagen molecule. One consequence of the formation of such heterotypic fibrils may be the regulation of corneal fibril diameter, a condition essential for corneal transparency.
The regulation of collagen fibril, bundle, and lamella formation by the corneal fibroblasts, as well as the organization of these elements into an orthogonal stroma, was studied by transmission electron microscopy and high voltage electron microscopy. Transmission and high voltage electron microscopy of chick embryo corneas each demonstrated a series of unique extracellular compartments. Collagen fibrillogenesis occurred within small surface recesses. These small recesses usually contained between 5 and 12 collagen fibrils with typically mature diameters and constant intrafibrillar spacing. The lateral fusion of the recesses resulted in larger recesses and consequent formation of prominent cell surface foldings. Within these surface foldings, bundles that contained 50-100 collagen fibrils were formed. The surface foldings continued to fuse and the cell surface retracted, forming large surface-associated compartments in which bundles coalesced to form lamellae. High voltage electron microscopy of 0.5 micron sections cut parallel to the corneal surface revealed that the corneal fibroblasts and their processes had two major axes at approximately right angles to one another. The surface compartments involved in the production of the corneal stroma were aligned along the fibroblast axes and the orthogonality of the cell was in register with that of the extracellular matrix. In this manner, corneal fibroblasts formed collagen fibrils, bundles, and lamellae within a controlled environment and thereby determined the architecture of the corneal stroma by the configuration of the cell and its associated compartments.
Decorin is a member of the expanding group of widely distributed small leucine-rich proteoglycans that are expected to play important functions in tissue assembly. We report that mice harboring a targeted disruption of the decorin gene are viable but have fragile skin with markedly reduced tensile strength. Ultrastructural analysis revealed abnormal collagen morphology in skin and tendon, with coarser and irregular fiber outlines. Quantitative scanning transmission EM of individual collagen fibrils showed abrupt increases and decreases in mass along their axes. thereby accounting for the irregular outlines and size variability observed in cross-sections. The data indicate uncontrolled lateral fusion of collagen fibrils in the decorindeficient mice and provide an explanation for the reduced tensile strength of the skin. These findings demonstrate a fundamental role for decorin in regulating collagen fiber formation in vivo.
Decorin belongs to a family of secreted, small, leucine-rich proteoglycans that affect matrix assembly and cellular growth. Ectopic expression of decorin proteoglycan or protein core as a mutated form lacking any glycosaminoglycan side chains induced growth suppression in neoplastic cells of various histogenetic origins, including tumor cells derived from gastrointestinal, genital, skeletal, cutaneous, or bone marrow tissues. Exogenously added recombinant decorin also suppressed overall growth of the parental cell lines. In all stably-transfected clones, growth retardation was specifically associated with induction of the potent cyclin-dependent kinase inhibitor p21, but not p27, and subsequent translocation of p21 protein into the nuclei of decorin-expressing cells. This led to a greater proportion of the cells arrested in G1 phase of the cell cycle. These changes were independent of functional p53 or retinoblastoma protein. De novo expression of decorin in HCT116 human colon carcinoma cells harboring a disrupted p21 gene failed to induce growth suppression, in contrast to the wild-type cells in which p21 and growth arrest could be induced. These findings indicate that ectopic production of decorin protein core can retard the growth of a variety of tumor cells and that endogenous p21 is a required downstream effector of this biological axis.
Bovine cornea contains three unique keratan sulfate proteoglycans (KSPGs), of which two (lumican and keratocan) have been characterized using molecular cloning. The gene for the third protein (KSPG25) has not been identified. This study examined the relationship between the KSPG25 protein and the gene for osteoglycin, a 12-kDa bone glycoprotein. The N-terminal amino acid sequence of KSPG25 occurs in osteoglycin cDNA cloned from bovine cornea. The osteoglycin amino acid sequence makes up the C-terminal 47% of the deduced sequence of the KSPG25 protein. Antibodies to osteoglycin reacted with intact corneal KSPG, with KSPG25 protein, and with a 36-kDa protein, distinct from lumican and keratocan. KSPG25-related proteins, not modified with keratan sulfate, were also detected in several connective tissues. Northern blot analysis showed mRNA transcripts of 2.4, 2.5, and 2.6 kilobases in numerous tissues with the 2.4-kilobase transcript enriched in ocular tissues. Ribonuclease protection analysis detected several protected KSPG25 mRNA fragments, suggesting alternate splicing of KSPG25 transcripts. We conclude that the full-length translation product of the gene producing osteoglycin is a corneal keratan sulfate proteoglycan, also present in many non-corneal tissues without keratan sulfate chains. The multiple size protein products of this gene appear to result from in situ proteolytic processing and/or alternative splicing of mRNA. The name mimecan is proposed for this gene and its products.
Decorin, a small leucine-rich proteoglycan, is capable of suppressing the growth of various tumor cell lines when expressed ectopically. In this report, we investigated the biochemical mechanism by which decorin inhibits cell cycle progression. In A431 squamous carcinoma cells, decorin proteoglycan or protein core induced a marked growth suppression, when either exogenously added or endogenously produced by a transgene. Decorin caused rapid phosphorylation of the EGF receptor and a concurrent activation of mitogen-activated protein (MAP) kinase signal pathway. This led to a protracted induction of endogenous p21, a potent inhibitor of cyclin-dependent kinases, and ultimate cell cycle arrest. Biglycan, a related proteoglycan, had no effect. Moreover, decorin activated the EGF receptor/MAP kinase/ p21 axis in cell lines of various histogenetic backgrounds. These results provide the first evidence that EGF and decorin converge functionally to regulate the cell cycle through activation of a common pathway which ultimately leads to growth suppression.
Lumican, a prototypic leucine-rich proteoglycan with keratan sulfate side chains, is a major component of the cornea, dermal, and muscle connective tissues. Mice homozygous for a null mutation in lumican display skin laxity and fragility resembling certain types of Ehlers-Danlos syndrome. In addition, the mutant mice develop bilateral corneal opacification. The underlying connective tissue defect in the homozygous mutants is deregulated growth of collagen fibrils with a significant proportion of abnormally thick collagen fibrils in the skin and cornea as indicated by transmission electron microscopy. A highly organized and regularly spaced collagen fibril matrix typical of the normal cornea is also missing in these mutant mice. This study establishes a crucial role for lumican in the regulation of collagen assembly into fibrils in various connective tissues. Most importantly, these results provide a definitive link between a necessity for lumican in the development of a highly organized collagenous matrix and corneal transparency.
To determine the effect of serum on morphology, growth, and proteoglycan synthesis by primary cultures of collagenase-isolated bovine keratocytes.
Keratocytes were isolated from bovine corneas using sequential collagenase digestion and cultured in Dulbecco's modified Eagle's medium (DMEM), with and without fetal bovine serum (FBS). Proteoglycans synthesized by the cells in culture and by keratocytes in intact cornea culture were metabolically radiolabeled with 35SO4. The proteoglycans were characterized by their sensitivity to keratanase, chondroitinase ABC, and heparatinase and by their size on Superose 6 HR. Cell number was determined by measuring DNA content of the culture dishes.
Keratocytes cultured in 10% FBS proliferated, appeared fibroblastic, and synthesized only 9% of the total glycosaminoglycan as keratan sulfate (KS), whereas cells in serum-free media were quiescent, appeared dendritic, and synthesized 47% KS, a value similar to the 45% KS for corneas radiolabeled overnight in organ culture. This increased proportion of KS synthesis in serum-free media was caused by a moderate increase in KS synthesis combined with a substantial decrease in chondroitin sulfate (CS) synthesis. Fractionation on Superose 6 High Resolution showed the size and relative amounts of the CS- and KS-containing proteoglycans synthesized by keratocytes in serum-free media also more closely resembled that of keratocytes in corneas in organ culture than keratocytes in media containing serum.
A comparison of proteoglycan synthesis and cell morphology between keratocytes in corneas in organ culture and in cell culture indicates that keratocytes maintain a more native biosynthetic phenotype and appearance when cultured in serum-free media. These results also suggest that culturing in the presence of serum fundamentally alters the keratocyte phenotype to an activated cell, mimicking certain changes observed during wound healing.
Lumican regulates collagenous matrix assembly as a keratan sulfate proteoglycan in the cornea and is also present in the connective tissues of other organs and embryonic corneal stroma as a glycoprotein. In normal unwounded cornea, lumican is expressed by stromal keratocytes. Our data show that injured mouse corneal epithelium ectopically and transiently expresses lumican during the early phase of wound healing, suggesting a potential lumican functionality unrelated to regulation of collagen fibrillogenesis, e. g. modulation of epithelial cell adhesion or migration. An anti-lumican antibody was found to retard corneal epithelial wound healing in cultured mouse eyes. Healing of a corneal epithelial injury in Lum(-/-) mice was significantly delayed compared with Lum(+/-) mice. These observations indicate that lumican expressed in injured epithelium may modulate cell behavior such as adhesion or migration, thus contributing to corneal epithelial wound healing.
Gene targeted lumican-null mutants (lum(tm1sc)/lum(tm1sc)) have cloudy corneas with abnormally thick collagen fibrils. The purpose of the present study was to analyze the loss of transparency quantitatively and to define the associated corneal collagen fibril and stromal defects.
Backscattering of light, a function of corneal haze and opacification, was determined regionally using in vivo confocal microscopy in lumican-deficient and wild-type control mice. Fibril organization and structure were analyzed using transmission electron microscopy. Biochemical approaches were used to quantify glycosaminoglycan contents. Lumican distribution in the cornea was elucidated immunohistochemically. RESULTS; Compared with control stromas, lumican-deficient stromas displayed a threefold increase in backscattered light with maximal increase confined to the posterior stroma. Confocal microscopy through-focusing (CMTF) measurement profiles also indicated a 40% reduction in stromal thickness in the lumican-null mice. Transmission electron microscopy indicated significant collagen fibril abnormalities in the posterior stroma, with the anterior stroma remaining relatively unremarkable. The lumican-deficient posterior stroma displayed a pronounced increase in fibril diameter, large fibril aggregates, altered fibril packing, and poor lamellar organization. Immunostaining of wild-type corneas demonstrated high concentrations of lumican in the posterior stroma. Biochemical assessment of keratan sulfate (KS) content of whole eyes revealed a 25% reduction in KS content in the lumican-deficient mice.
The structural defects and maximum backscattering of light clearly localized to the posterior stroma of lumican-deficient mice. In normal mice, an enrichment of lumican was observed in the posterior stroma compared with that in the anterior stroma. Taken together, these observations indicate a key role for lumican in the posterior stroma in maintaining normal fibril architecture, most likely by regulating fibril assembly and maintaining optimal KS content required for transparency.
Collagen fibrillogenesis is finely regulated during development of tissue-specific extracellular matrices. The role(s) of a leucine-rich repeat protein subfamily in the regulation of fibrillogenesis during tendon development were defined. Lumican-, fibromodulin-, and double-deficient mice demonstrated disruptions in fibrillogenesis. With development, the amount of lumican decreases to barely detectable levels while fibromodulin increases significantly, and these changing patterns may regulate this process. Electron microscopic analysis demonstrated structural abnormalities in the fibrils and alterations in the progression through different assembly steps. In lumican-deficient tendons, alterations were observed early and the mature tendon was nearly normal. Fibromodulin-deficient tendons were comparable with the lumican-null in early developmental periods and acquired a severe phenotype by maturation. The double-deficient mice had a phenotype that was additive early and comparable with the fibromodulin-deficient mice at maturation. Therefore, lumican and fibromodulin both influence initial assembly of intermediates and the entry into fibril growth, while fibromodulin facilitates the progression through growth steps leading to mature fibrils. The observed increased ratio of fibromodulin to lumican and a competition for the same binding site could mediate these transitions. These studies indicate that lumican and fibromodulin have different developmental stage and leucine-rich repeat protein specific functions in the regulation of fibrillogenesis.
To analyse the human corneal stroma in extreme hydration to discover if its structure is responsible for corneal stability.
Corneas in several hydration states were used: postmortem control corneas (PM; n=3), corneas left for 1 day in phosphate buffered saline (PBS; n=4), and corneas left for 1 day (n=4), 2 days (n=4), 3 days (n=2), and 4 days (n=4) in deionised water. All corneas were fixed under standardised conditions and processed for light and electron microscopy. In addition, two fresh corneas from the operating theatre were studied which were processed 6 months after storage in sodium cacodylate buffer.
After 1 day in deionised water maximal stromal swelling was reached which did not change up to 4 days. The stroma of deionised water corneas (1400 microm) was much thicker than that of PBS corneas (650 microm) and PM corneas (450 microm). Deionised water treatment led to disappearance of all keratocytes leaving only remnants of nuclei and large interlamellar spaces. In these specimens the distance between the collagen fibres had increased significantly, but the diameter of the collagen fibres did not seem to be affected. A remarkable observation was that the most anterior part of the stroma (100-120 microm) in all deionised water specimens and those stored for 6 months in buffer was not swollen, indicating that the tightly interwoven anterior lamellae are resistant to extreme non-physiological hydration states.
The rigidity of the most anterior part of the corneal stroma in extreme hydration states points to an important role in maintenance of corneal curvature. Since a large part of this rigid anterior part of the stroma is either removed (PRK) or intersected (LASIK), it is possible that in the long run patients who underwent refractive surgery may be confronted with optical problems.
The small-diameter fibrils of the chick corneal stroma are heterotypic, composed of both collagen types I and V. This tissue has a high concentration of type V collagen relative to other type I-containing tissues with larger-diameter fibrils, suggesting that heterotypic interactions may have a regulatory role in the control of fibril diameter. The interactions of collagen types I and V were studied using an in vitro self-assembly system. Collagens were purified from lathyritic chick embryos in the presence of protease inhibitors. The type V collagen preparations contained higher molecular weight forms of the alpha 1(V) and alpha 2(V) chains constituting 60–70% of the total. Rotary-shadow electron micrographs showed a persistence of a small, pepsin-sensitive terminal region in an amount consistent with that seen by electrophoresis. In vitro, this purified type V collagen formed thin fibrils with no apparent periodicity, while type I collagen fibrils had a broad distribution of large diameters. However, when type I collagen was mixed with increasing amounts of type V collagen a progressive and significant decrease in both the mean fibril diameter and the variance was observed for D periodic fibrils. The amino-terminal domain of the type V collagen molecule was required for this regulatory effect and in its absence little diameter reducing activity was observed. Electron microscopy using collagen type-specific monoclonal antibodies demonstrated that the fibrils formed were heterotypic, containing both collagen types I and V. These data indicate that the interaction of type V with type I collagen is one mechanism modulating fibril diameter and is at least partially responsible for the regulation of collagen fibril formation.
AIM
To analyse the human corneal stroma in extreme hydration to discover if its structure is responsible for corneal stability.
METHODS
Corneas in several hydration states were used: postmortem control corneas (PM; n=3), corneas left for 1 day in phosphate buffered saline (PBS; n=4), and corneas left for 1 day (n=4), 2 days (n=4), 3 days (n=2), and 4 days (n=4) in deionised water. All corneas were fixed under standardised conditions and processed for light and electron microscopy. In addition, two fresh corneas from the operating theatre were studied which were processed 6 months after storage in sodium cacodylate buffer.
RESULTS
After 1 day in deionised water maximal stromal swelling was reached which did not change up to 4 days. The stroma of deionised water corneas (1400 μm) was much thicker than that of PBS corneas (650 μm) and PM corneas (450 μm). Deionised water treatment led to disappearance of all keratocytes leaving only remnants of nuclei and large interlamellar spaces. In these specimens the distance between the collagen fibres had increased significantly, but the diameter of the collagen fibres did not seem to be affected. A remarkable observation was that the most anterior part of the stroma (100–120 μm) in all deionised water specimens and those stored for 6 months in buffer was not swollen, indicating that the tightly interwoven anterior lamellae are resistant to extreme non-physiological hydration states.
CONCLUSIONS
The rigidity of the most anterior part of the corneal stroma in extreme hydration states points to an important role in maintenance of corneal curvature. Since a large part of this rigid anterior part of the stroma is either removed (PRK) or intersected (LASIK), it is possible that in the long run patients who underwent refractive surgery may be confronted with optical problems.
purpose. Identification of mutations in the CHST6 gene in 15 patients from 11 unrelated families affected with recessive macular corneal dystrophy (MCD).
methods. Genomic DNA was extracted from peripheral blood leukocytes of the affected patients and their healthy family members, and the mutational status of the CHST6 gene was determined for each patient by a PCR-sequencing approach. Serum concentrations of antigenic keratan sulfate for each proband were determined by ELISA.
results. ELISA indicated that all affected patients, except one, were of MCD type I or IA. Fourteen distinct mutations were identified within the CHST6 coding region: 2 nonsense, 2 frameshift, and 10 missense. Of these, 12 were novel, and a nonsense mutation in the homozygous state is reported for the first time.
conclusions. These molecular results in French patients with MCD combined with those reported in previous studies indicated CHST6 mutational heterogeneity. The characterization herein of nonsense mutations is in keeping with the fact that MCD results from loss of function of the CHST6 protein product.
The techniques of exploratory data analysis include a resistant rule for identifying possible outliers in univariate data. Using the lower and upper fourths, FL and FU (approximate quartiles), it labels as “outside” any observations below FL − 1.5(FU — FL) or above FU + 1.5(FU — FL). For example, in the ordered sample −5, −2, 0, 1, 8, FL = −2 and FU = 1, so any observation below −6.5 or above 5.5 is outside. Thus the rule labels 8 as outside. Some related rules also use cutoffs of the form FL — k(FU — FL) and FU + k(FU — FL). This approach avoids the need to specify the number of possible outliers in advance; as long as they are not too numerous, any outliers do not affect the location of the cutoffs.To describe the performance of these rules, we define the some-outside rate per sample as the probability that a sample will contain one or more outside observations. Its complement is the all-inside rate per sample. We also define the outside rate per observation as the average fraction of outside observations. For Gaussian data the population all-inside rate per sample (0) and the population outside rate per observation (.7%) substantially understate the corresponding small-sample values. Simulation studies using Gaussian samples with n between 5 and 300 yield detailed information on the resistant rules. The main resistant rule (k = 1.5) has an all-inside rate per sample between 67% and 86% for 5 ≤n ≤ 20, and corresponding estimates of its outside rate per observation range from 8.6% to 1.7%.Both characteristics vary with n in ways that lead to good empirical approximations. Because of the way in which the fourths are defined, the sample sizes separate into four classes, according to whether dividing n by 4 leaves a remainder of 0, 1, 2, or 3. Within these four classes the all-inside rate per sample shows a roughly linear decrease with n over the range 9 ≤ n ≤ 50, and the outside rate per observation decreases linearly in 1/n for n ≥ 9.A more theoretical approximation for the all-inside rate per sample works with the order statistics X(1) ≤ … ≤ X(n). In this notation the fourths are X(f) and X(n + 1 — f) with f = ½[(n + 3)/2], where [·] is the greatest-integer function. A sample has no observations outside whenever {X(f)−X(1)}/{X(n+1-f)−X(f)}≤k and {X(n)−X(n+1-f)}/{X(n+1-f)−X(f)}≤k. We first approximate the numerators and denominator in these ratios by constant multiples of chi-squared random variables with the same mean and variance. We then approximate the logarithm of each ratio by a Gaussian random variable, and we calculate the correlation between these variables from the fact that the ratios have the same denominator. Finally, a bivariate Gaussian probability calculation yields the approximate all-inside rate per sample. The error of the result relative to the simulation estimate is typically from 1% to 2% for 5 ≤ n ≤ 50.To provide an indication of how the two rates behave in alternative “null” situations, the simulation studies included samples from five heavier-tailed members of the family of h distributions. For a given sample size, the all-inside rate per sample decreases as the tails become heavier, and the outside rate per observation increases.
A previous study examined the performance of a standard rule from Exploratory Data Analysis, which uses the sample fourths, FL and FU, and labels as “outside” any observations below FL – k(FU – FL) or above FU + k(FU – FL), customarily with k = 1.5. In terms of the order statistics X(1) ≤ X(2) ≤ X(n) the standard definition of the fourths is FL = X(f) and FU = X(n + 1 − f), where f = ½[(n + 3)/2] and [·] denotes the greatest-integer function. The results of that study suggest that finer interpolation for the fourths might yield smoother behavior in the face of varying sample size. In this article we show that using fi = n/4 + (5/12) to define the fourths produces the desired smoothness. Corresponding to a common definition of quartiles, fQ = n/4 + (1/4) leads to similar results. Instead of allowing the some-outside rate per sample (the probability that a sample contains one or more outside observations, analogous to the experimentwise error rate in simultaneous inference) to vary, some users may prefer to maintain it at .10 or .05 for Gaussian data and vary k accordingly. We obtain such values of k at selected sample sizes n ≤ 300.
The water sorptive and retentive capacities of three corneal proteoglycans with different keratan sulfate/chondroitin-4-sulfate compositions were investigated. The calcium salt of a predominantly keratan sulfate containing proteoglycan had hydration properties similar to that of calcium keratan sulfate. The proteoglycan containing predominantly calcium chondroitin-4-sulfate side chains sorbed water to a greater extent than pure calcium chondroitin-4-sulfate but its retentive power was somewhat less. The proteoglycan containing about twice as much keratan sulfate as chondroitin-4-sulfate, on a dissaccharidic molar basis and had hydration properties which were closer to the behavior of chondroitin-4-sulfate than keratan sulfate. The results are discussed in terms of structure and polymer interaction in the proteoglycan matrices.
This article on the development of the vertebrate cornea is divided into five major parts as follows: the general outline of corneal morphogenesis includes the avian cornea, the primate cornea, and other vertebrates. The parts on the corneal epithelium covers secretory organelles and secretion of stroma, the structure and fate of the primary corneal stroma, and epithelial stratification and innervation. The third part, the corneal endothelium, includes the origin of the endothelium, secretory functions of the endothelium, and the origin of Descemet's Membrane. The corneal fibroblasts (keratocytes) include the mode of migration, secretory products of the fibroblasts and transparency, thyroxine, and stomal condensation. Lastly, the part on tissue interaction in the developing cornea covers inductive events during formation of the primary stroma, the capacity of epithelial cells for metaplasia, inductive events during the formation of the endothelium and tissue interaction in the definite cornea.
Excised bovine cornea were sectioned from epithelium to endothelium into 6 to 8 fractions. The glucosamine/galactosamine ratio steadily increases from epithelium to endothelium in all bovine corneas investigated. The glucosamine/galactosamine ratio reflects the keratan sulfate/chondroitin-4-sulfate ratio in the cornea. The significance of this topographic distribution is discussed in terms of the different hydration properties of proteoglycans containing predominantly keartan sulfate or chondroitin-4-sulfate chains.
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The small-diameter fibrils of the chick corneal stroma are heterotypic, composed of both collagen types I and V. This tissue has a high concentration of type V collagen relative to other type I-containing tissues with larger-diameter fibrils, suggesting that heterotypic interactions may have a regulatory role in the control of fibril diameter. The interactions of collagen types I and V were studied using an in vitro self-assembly system. Collagens were purified from lathyritic chick embryos in the presence of protease inhibitors. The type V collagen preparations contained higher molecular weight forms of the alpha 1(V) and alpha 2(V) chains constituting 60-70% of the total. Rotary-shadow electron micrographs showed a persistence of a small, pepsin-sensitive terminal region in an amount consistent with that seen by electrophoresis. In vitro, this purified type V collagen formed thin fibrils with no apparent periodicity, while type I collagen fibrils had a broad distribution of large diameters. However, when type I collagen was mixed with increasing amounts of type V collagen a progressive and significant decrease in both the mean fibril diameter and the variance was observed for D periodic fibrils. The amino-terminal domain of the type V collagen molecule was required for this regulatory effect and in its absence little diameter reducing activity was observed. Electron microscopy using collagen type-specific monoclonal antibodies demonstrated that the fibrils formed were heterotypic, containing both collagen types I and V. These data indicate that the interaction of type V with type I collagen is one mechanism modulating fibril diameter and is at least partially responsible for the regulation of collagen fibril formation.
Twenty-six bovine corneas, 30 micron thick, were sectioned perpendicular to the visual axis. Sections, weighing 10-20 mg, were analyzed for total water content by thermogravimetric analysis and for freezable water content by differential scanning calorimetry. The total water content as well as the free (freezable) water content increase, while the bound (nonfreezable) water content decreases in progressing from epithelium to endothelium. These results are correlated with the distribution of proteoglycans and their water sorptive and retentive capacity in bovine cornea.
CELLS of both the reproductive and functional compartments of oral and
cutaneous epithelia are at present indiscriminately called
``keratinocytes''. The two types of cells, however, differ markedly in
function and fate, and it is therefore logical, as well as useful, to
distinguish them by different terms. It is therefore proposed to
introduce the term ``keratoblast'' to denote a cell in the reproductive
compartment of these tissues, and thus to distinguish dividing cells
from those that produce keratin.
The small dermatan sulphate proteoglycan of bovine tendon demonstrated a unique ability to inhibit fibrillogenesis of both type I and type II collagen from bovine tendon and cartilage respectively in an assay performed in vitro. None of the other proteoglycan populations from cartilage, tendon or aorta, even those similar in size and chemical structure, had this effect. Alkali treatment of the small proteoglycan of tendon eliminated its ability to inhibit fibrillogenesis, whereas chondroitinase digestion did not. This indicates that its interaction with collagen depends on the core protein. Fibrillogenesis of pepsin-digested collagens was affected similarly, indicating that interaction with the collagen telopeptides is not involved. The results suggest that interactions between collagen and proteoglycans may be quite specific both for the type of proteoglycan and its tissue of origin.
Corneal specimens obtained during surgery from patients with macular corneal dystrophy and obtained at autopsy from control eyes were incubated in a medium containing radioactive precursors of glycoproteins and proteoglycans. Biosynthetically radiolabeled material was extracted and characterized by using molecular sieve chromatography and specific enzymes. Cells in control corneas synthesized both a chondroitin sulfate proteoglycan and a keratan sulfate proteoglycan similar to those present in monkey and bovine corneas. Cells in macular corneas synthesized a normal chondroitin sulfate proteoglycan but did not synthesize either keratan sulfate or a mature keratan sulfate proteoglycan. Instead, macular corneas synthesized a glycoprotein with unusually large oligosaccharide side chains. This glycoprotein was not detected in normal corneas and is slightly smaller than normal keratan sulfate proteoglycan. The failure to synthesize a mature keratan sulfate proteoglycan may produce corneal opacity and result in blindness. Because of evidence indicating that the corneal keratan sulfate proteoglycan is normally synthesized through a glycoprotein intermediate [Hart, G. W. & Lennarz, W. (1978) J. Biol. Chem. 253-5795-5801], macular corneal dystrophy may be a defect in glycoprotein processing.
Corneal scars generated in rabbits by penetrating wounds are initially opaque but become transparent within a year. Previous studies have shown that the corneal stroma consists of proteoglycans and collagen fibrils spaced at regular intervals and that the interfibrillar spaces, the presumed location of proteoglycans, are abnormally large in opaque scars. In the present study, the size and glycosaminoglycan composition of the corneal stromal proteoglycans were determined in corneal scars during the restoration of transparency. The results showed that initially opaque scars which contained the large interfibrillar spaces also contained unusually large chondroitin sulfate proteoglycans with glycosaminoglycan side chains of normal size. These opaque scars also lacked the keratan sulfate proteoglycan but did contain hyaluronic acid. In the 1-year-old scars there was a restoration of normal interfibrillar spacing, and a return to corneal stromal proteoglycans of normal size and composition. These correlations suggest that the corneal stromal proteoglycans may play a fundamental role in regulating corneal collagen fibril spacing.
The authors sought to discover whether there are differences in the degree of spatial order in the fibrillar ultrastructure between anterior and posterior stroma.
Human corneas were obtained from eye bank eyes. Although they had been classified as normal, some swelling remained after 3 hours of deturgescence. Freshly excised, unswollen rabbit corneas also were used. Image analysis methods were applied to transmission electron micrographs of the anterior, middle, and posterior stroma of these corneas to determine the positions and radii of fibrils, the fraction of total area occupied by fibrils, and the fibril number density. Results were used to calculate the interference factor that appears in the direct summation of the fields for light scattering theory and to estimate the total scattering cross-section per fibril. The interference factor is a measure of the spatial order in the positions and sizes of the fibrils.
Electron micrographs showed anterior-posterior variations in size and number density of fibrils. The interference factor at wavelengths of visible light was lower in posterior stroma than in anterior stroma for humans and rabbits. In some instances in humans, the anterior interference factor was characteristic of mildly swollen cornea. When averaged for the electron micrographs analyzed, the anterior stroma was predicted to scatter approximately twice as much light per unit depth as the posterior stroma in humans (at any given wavelength) and approximately three times as much in rabbits.
Calculations of the interference factor showed that there were differences in the anterior-posterior spatial ordering of fibrils. In human corneas, the differences could have been caused by intrinsic in vivo differences between anterior and posterior stroma; however, possible anterior-posterior variations in swelling between the two regions in vitro also could have affected the results.
Corneal transparency is dependent on the size and arrangement of collagen fibrils within the corneal stroma. The corneal stroma is composed primarily of collagen type I fibrils and two proteoglycans; one with chondroitin/dermatan sulfate side-chains (decorin) and one with keratan sulfate side-chains (lumican). We investigated the effects of the corneal proteoglycans on corneal collagen fibrillogenesis, utilizing an in vitro assay for fibril formation. Collagen was extracted from bovine corneal stromas with 0·1 M acetic acid and monomers purified by NaCl precipitation, Decorin and lumican were extracted from bovine cornea/stroma with either 0·7 M NaCl or 4 M guanidine HCl and purified by DEAE and Sepharose CL-4B chromatography. Decorin and lumican from both extracts inhibited the rate of collagen fibrillogenesis and the development of turbidity in fibrillogenesis samples. Furthermore, the core proteins of decorin and lumican were shown to be as effective as the intact proteoglycans in inhibiting fibrillogenesis. The decorin core protein isolated from the 0·7 M NaCl extract was determined to be a 20 kDa fragment which lacks the C-terminal half of the core protein. This fragment was ≈ 1/36 as effective in inhibiting fibrillogenesis as intact decorin isolated from guanidine extracts. This suggests that the C-terminal half of the decorin core plays an important role in the interaction of this proteoglycan with collagen.
The cornea, dermis and tendon have extracellular matrix architectures with differences in fibril diameter, packing and organization. An early step in fibril assembly is the formation of a striated fibril of discrete length (segment). Fibril segments were isolated from developing chicken cornea, dermis and tendon by physical disruption and the structure characterized. In all three tissues, intact but relatively short fibril lengths were isolated. These segments were asymmetric, having long (alpha) and short (beta) tapered ends. They were also centrosymmetric with respect to molecular packing. Segments isolated from 12- to 16-day corneas, dermis and tendons had identical structures, but their lengths and diameters were distinct. We propose that the increase in length is, at least in part, the result of lateral associations of adjacent segments. In the developing tendon, there is a rapid increase in length and diameter between day 16 and 17, while in the dermis the increase is more linear with respect to time. In the cornea, the fibril segments grow longer, but their diameters remain constant. Disruption of corneas in phosphate-buffered saline yielded larger diameter segments than seen in situ, while tendon or dermis maintained tissue-specific diameters. When corneas were disrupted in buffers that stabilized the water layer associated with the collagen molecules or containing the corneal proteoglycans, then tissue-specific diameters were maintained. These data suggest differences in the stabilization of segments during growth in tissues where diameter increases versus those where diameter remains constant, and this may be related to collagen-proteoglycan interactions.
In mammals, tissue damage is usually repaired by activation of a fibrotic response which saves the life of the organism, but which can never restore function to the damaged organ. In addition, fibrotic responses form the basis for diverse pathologies, including many that occur in the eye. It is intriguing, therefore, to observe the occasional circumstances in which repair in mammals appears to take on a regenerative character, such as during fetal wound healing or in certain types of corneal wounds. The thesis of this chapter is that the choice between regeneration or fibrosis lies in the control of fibroblast phenotype. The cornea of the eye has several features which make it a particularly useful model for the study of fibroblast phenotype. Studies discussed herein, identify failure to activate the transcription factor NF-kappaB as a control mechanism for inhibiting fibroblast activation in the cornea. Evidence is further presented for the view that transition in fibroblast phenotype in repair tissue is not simply a matter of differential gene expression, but is a developmental event which reflects changes in the hard wiring of signalling pathways by which the cell responds to environmental input.
To determine whether temporal and spatial changes in the distribution of the long and short alternatively spliced variants of type XII collagen are associated with any specific morphogenetic events in pre- and postnatal development of the cornea and surrounding tissues.
The distribution of alternatively spliced variants of type XII collagen in fetal and newborn rabbit tissues was analyzed immunohistochemically using monoclonal antibodies that recognize either only the long form or both the short and the long forms of type XII collagen.
During early fetal development of the cornea in rabbit (days 14 -17), the short form of type XII collagen was detected in the corneal stroma, the sclera, and the stroma in the rudimentary eyelid folds, whereas the long form was present only in the sclera. The long form was first evident in the cornea at day 24 but only in the posterior stroma. At later stages of prenatal development, the distribution of the long variant gradually extended toward the anterior stroma and in the newborn rabbit, the long variant was distributed throughout the entire stroma. However, in the eyelid, although the short form was present along the entire subepidermal regions both during fetal and neonatal development, the long form was transiently expressed between days 21 and 24 and was restricted to the subepidermal regions at the junction of the opposing eyelids. The long form of type XII collagen was first detectable in the basal epithelial cells and in its basement membrane (BM) at day 12 after birth, just before the opening of the eyelids. It continued to be present in the corneal BM zone in the adult rabbit but was not present in the limbal or conjunctival BM zone.
The expression and distribution of the alternatively spliced forms of type XII collagen are developmentally and differentially regulated in the cornea, the sclera, and the eyelid. Although the short form is expressed in the stromal matrices of the cornea and surrounding tissues from early stages of corneal development, the appearance and distribution of the long variant form of type XII collagen coincide with the pattern of stromal condensation. Its first appearance in the corneal epithelial BM precedes the eyelid opening by 1 to 2 days, possibly suggesting that it may be involved in the tighter anchoring of the corneal epithelium to the underlying tissue or in promoting stromal condensation to assist in the separation of the corneal epithelium from the juxtaposed palpebral conjunctival epithelium of the eyelid.
The corneal proteoglycans belong to the Leu-rich proteoglycan (LRP) gene family and contain chondroitin/dermatan (CS/DS) or keratan sulfate (KS) chains. These proteoglycans play a critical role in generating and maintaining a transparent matrix within the corneal stroma. Decorin which has CS/DS chains and lumican which has KS chains, were first to be identified in the cornea. Two other corneal KS proteoglycans (KSPGs), keratocan and osteoglycin/mimecan were recently identified in bovine corneas. We cloned and sequenced chick osteoglycin/mimecan and found it to contain a stretch of 60 amino acids that showed no identity to the presumed mammalian homolog. The 177 base pair DNA coding for this unique sequence shows 47% identity to an 189 base pair sequence between exons 4 and 5 of the bovine osteoglycin/mimecan gene. This indicates that this cDNA represents an alternatively spliced form of osteoglycin/mimecan containing a unique N-terminal sequence.
Fibromodulin and lumican are closely related members of the extracellular matrix leucine-rich repeat glycoprotein/proteoglycan family. Similar to decorin, another member of this protein family, they bind to fibrillar collagens and function in the assembly of the collagen network in connective tissues. We have studied the binding of recombinant fibromodulin, lumican and decorin, expressed in mammalian cells, to collagen type I. Using a collagen fibril formation/sedimentation assay we show that fibromodulin inhibits the binding of lumican, and vice versa. Fibromodulin and lumican do not affect the binding of decorin to collagen, nor does decorin inhibit the binding of fibromodulin or lumican. Binding competition experiments and Scatchard plot analysis indicate that fibromodulin binds to collagen type I with higher affinity than lumican.
Type V collagen is a quantitatively minor fibrillar collagen with a broad tissue distribution. The most common type V collagen isoform is alpha1(V)(2) alpha2(V) found in cornea. However, other isoforms exist, including an [alpha1(V)alpha2(V)alpha3(V)] form, an alpha1(V)(3) homotrimer and hybrid type V/XI forms. The functional role and fibrillar organization of these isoforms is not understood. In the cornea, type V collagen has a key role in the regulation of initial fibril assembly. Type I and type V collagen co-assemble into heterotypic fibrils. The entire triple-helical domain of the type V collagen molecules is buried within the fibril and type I collagen molecules are present along the fibril surface. The retained NH(2)-terminal domains of the type V collagen are exposed at the surface, extending outward through the gap zones. The molecular model of the NH(2)-terminal domain indicates that the short alpha helical region is a flexible hinge-like region allowing the peptide to project away from the major axis of the molecule; the short triple-helical regions serve as an extension through the hole zone, placing the tyrosine-rich domain at the surface. The assembly of early, immature fibril intermediates (segments) is regulated by the NH(2)-terminal domain of type V collagen. These NH(2)-terminal domains alter accretion of collagen molecules onto fibrils and therefore lateral growth. A critical density would favor the initiation of new fibrils rather than the continued growth of existing fibrils. Other type V collagen isoforms are likely to have an important role in non-cornea tissues. This role may be mediated by supramolecular aggregates different from those in the corneal stroma or by an alteration of the interactions mediated by tissue-specific type V collagen domains generated by different isoforms or aggregate structures. Presumably, the aggregate structure or specific domains are involved in the regionalization of fibril-associated macromolecules necessary for the tissue-specific regulation of later fibril growth and matrix assembly stages.
The last 5 years have seen a marked increase in research on keratan sulfate (KS) and a concomitant increase in our understanding of the range of molecules that carry this adaptable polysaccharide. More than 15 KS-linked proteins have been identified and many of the genes encoding these have been cloned. KS-containing molecules have been identified in numerous epithelial and neural tissues in which KS expression responds to embryonic development, physiological variations, and to wound healing. A corneal cell culture system has been developed in which long-term KS biosynthesis is maintained. Progress has been made toward identification of the glycosyl- and sulfotransferases responsible for KS biosynthesis. A mouse knockout of a corneal KS-proteoglycan has provided the first experimental support for the role of KS in corneal transparency. Evidence has also been presented supporting functional roles of KS in cellular recognition of protein ligands, axonal guidance, cell motility, and in embryo implantation. These findings have served to expand the concept of what keratan sulfate is and the potential roles it may play in the cellular biology of diverse tissues.
Many solutions have been used to investigate the swelling properties of the mammalian corneal stroma but few of the solutions resemble the expected extracellular matrix fluid of the corneal stroma, and little information is available on whether incubation ex vivo causes significant changes in the gross composition of the stroma. From quality-selected recent post-mortem eyes of adult cattle, stroma preparations were cut from the central part of the cornea. The time-dependent changes in wet mass were assessed over 9 h at 37 degrees C, and the preparations then dried. Various solutions of known pH (6.88-8.32) and osmolality (<50-327 mosmol/kg) were used, and were assayed for protein and proteoglycan after the incubation. The rates and extent of stromal swelling were lowest in a glucose-supplemented mixed salts solution containing 35 mM bicarbonate (0.5% CO2) solution, marginally greater in a mixed salts solution containing 35 mM bicarbonate (5% CO2) or similar non-bicarbonate mixed salts solutions (including BSS), and progressively greater in phosphate-buffered saline (PBS), various phosphate buffers (10-67 mM) and saline solutions (0.025-1%), and greatest in water. The initial rates of swelling ranged from 44 to 451 mg/h and the secondary rates from 9 to 106 mg/h. In all solutions, protein and proteoglycans were detected, but these ranged from around 1 to 10% of the samples with the bicarbonate-buffered solutions, to around 30% with the use of some phosphate buffers or saline.
The corneas of mice homozygous for a null mutation in lumican, a keratan sulfate-containing proteoglycan, are not as clear as normal. In the present study, mutant corneas were examined by synchrotron x-ray diffraction to see what structural changes might lie behind the loss of transparency.
X-ray diffraction patterns were obtained from the corneas of 6-month-old and 2-month-old lumican-null and wild-type mice. Measured in each cornea were the average collagen fibril diameter, average collagen fibril spacing, and the level of order in the collagen array.
The x-ray reflection arising from regularly packed collagen was well-defined on all x-ray patterns from 6-month-old wild-type corneas. Patterns from 6-month-old lumican-deficient corneas, however, contained interfibrillar reflections that were measurably more diffuse, a fact that points to a widespread alteration in the way the collagen fibrils are configured. The same distinction between mutant and wild-type corneas was also noted at 2-months of age. Average collagen fibril spacing was marginally higher in corneas of 6-month-old lumican-null mice than in corneas of normal animals. Unlike x-ray patterns from wild-type corneas, patterns from lumican-deficient corneas of both ages registered no measurable subsidiary x-ray reflection, evidence of a wider than normal range of fibril diameters.
The spatial arrangement of stromal collagen in the corneas of lumican-deficient mice is in disarray. There is also a considerable variation in the diameter of the hydrated collagen fibrils. These abnormalities, seen at 2 months as well as 6 months of age, probably contribute to the reduced transparency.