Robert D. Young

Cardiff University

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Matrix morphogenesis in cornea is mediated by the modification of keratan sulfate by GlcNAc 6- O -sulfotransferase

Proceedings of the National Academy of Sciences (2006)

Yasutaka Hayashida Tomoya O. Akama Nicola Beecher Philip N. Lewis Robert D. Young

Matrix assembly and homeostasis in collagen-rich tissues are mediated by interactions with proteoglycans (PGs) substituted with sulfated glycosaminoglycans (GAGs). The major GAG in cornea is keratan sulfate (KS), which is N-linked to one of three PG core proteins. To ascertain the importance of the carbohydrate chain sulfation step in KS functionality, we generated a strain of mice with a targeted gene deletion in Chst5, which encodes an N-acetylglucosamine-6-O-sulfotransferase that is integral to the sulfation of KS chains. Corneas of homozygous mutants were significantly thinner than those of WT or heterozygous mice. They lacked high-sulfated KS, but contained the core protein of the major corneal KSPG, lumican. Histochemically stained KSPGs coassociated with fibrillar collagen in WT corneas, but were not identified in the Chst5-null tissue. Conversely, abnormally large chondroitin sulfate/dermatan sulfate PG complexes were abundant throughout the Chst5-deficient cornea, indicating an alteration of controlled PG production in the mutant cornea. The corneal stroma of the Chst5-null mouse exhibited widespread structural alterations in collagen fibrillar architecture, including decreased interfibrillar spacing and a more spatially disorganized collagen array. The enzymatic sulfation of KS GAG chains is thus identified as a key requirement for PG biosynthesis and collagen matrix organization.

Keratan sulfate

Lumican

Dermatan sulfate

Chondroitin

Perlecan

10.1073/pnas.0605441103

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Citations (81)

Role of Decorin Core Protein in Collagen Organisation in Congenital Stromal Corneal Dystrophy (CSCD)

PLoS ONE (2016)

Christina S. Kamma‐Lorger Christian Pinali Juan Carlos Martínez Jon Harris Robert D. Young

The role of Decorin in organising the extracellular matrix was examined in normal human corneas and in corneas from patients with Congenital Stromal Corneal Dystrophy (CSCD). In CSCD, corneal clouding occurs due to a truncating mutation (c.967delT) in the decorin (DCN) gene. Normal human Decorin protein and the truncated one were reconstructed in silico using homology modelling techniques to explore structural changes in the diseased protein. Corneal CSCD specimens were also examined using 3-D electron tomography and Small Angle X-ray diffraction (SAXS), to image the collagen-proteoglycan arrangement and to quantify fibrillar diameters, respectively. Homology modelling showed that truncated Decorin had a different spatial geometry to the normal one, with the truncation removing a major part of the site that interacts with collagen, compromising its ability to bind effectively. Electron tomography showed regions of abnormal stroma, where collagen fibrils came together to form thicker fibrillar structures, showing that Decorin plays a key role in the maintenance of the order in the normal corneal extracellular matrix. Average diameter of individual fibrils throughout the thickness of the cornea however remained normal.

Corneal dystrophy

10.1371/journal.pone.0147948

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Citations (29)

Three-dimensional aspects of matrix assembly by cells in the developing cornea

Proceedings of the National Academy of Sciences (2014)

Robert D. Young Carlo Knupp Christian Pinali Kenneth M.Y. Png James R. Ralphs

Significance The cornea is a specialized connective tissue assembled as a remarkably ordered array of superimposed collagenous lamellae, and their component collagen fibrils, essential for optical transparency. Surprisingly, the mechanisms involved in deposition of this unique structure are still not fully understood. Here we have used correlative microscopy techniques, including innovative methods of serial block face scanning electron microscopy, to observe the sequence of corneal matrix formation in three-dimensional reconstructions of embryonic chick cornea. Our data show that corneal cells, keratocytes, exhibit long-range associations with collagen bundles in the developing matrix via an extended network of actin-rich tubular cytoplasmic protrusions, which we term keratopodia . Synchronized alignment of keratopodia and collagen is evident during the course of lamella formation.

Lamella (surface anatomy)

Matrix (chemical analysis)

Collagen fibril

10.1073/pnas.1313561110

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Citations (76)

Imaging pathology in archived cornea with Fuchs’ endothelial corneal dystrophy including tissue reprocessing for volume electron microscopy

Scientific Reports (2024)

Sayo Maeno Philip N. Lewis Robert D. Young Yoshinori Oie Kohji Nishida

Fuchs' endothelial corneal dystrophy (FECD) is a common sight-threatening condition characterised by pathological changes in the posterior cornea. Here we report observations by light, transmission and volume scanning electron microscopy on changes in the endothelium and matrix associated with the characteristic deformations of Descemet's membrane, termed guttae. Specimens were archived full-thickness human corneal tissue, removed during graft surgery, that had been fixed, stained and embedded by conventional processing methods for examination by transmission electron microscopy more than 40-years previously. Intact archived samples can be extremely valuable where, as with FECD, new cell-based methods of therapy now avoid excision of the full cornea thickness and any tissue excised is inferior for study. Volume electron microscopy, in particular serial block face scanning electron microscopy (SBF SEM), employing backscatter electron detection from resin-embedded specimens, has become an invaluable technique for 3D imaging of biological samples. However, archived specimens are normally considered unsuitable for imaging as conventional processing methods generate low backscatter electron yield. To overcome this for SBF SEM, we subjected epoxy resin-embedded specimens to de-plastination, then applied additional contrasting agents, uranyl acetate and lead acetate, prior to re-embedding. Selected regions of interest in the new resin blocks were examined in a scanning electron microscope equipped for SBF SEM and serial image datasets acquired. Enhanced contrast enabled 3D reconstruction of endothelium and guttae in Descemet's membrane over large tissue volumes.

Dystrophy

10.1038/s41598-024-82888-5

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Citations (0)

Generation of 3D lacrimal gland organoids from human pluripotent stem cells

Nature (2022)

Ryuhei Hayashi Toru Okubo Yuji Kudo Yuki Ishikawa Tsutomu Imaizumi

Organoid

10.1038/s41586-022-04613-4

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Citations (43)

The effect of bacteriochlorophyll derivative WST-D and near infrared light on the molecular and fibrillar architecture of the corneal stroma

Scientific Reports (2020)

Sally Hayes Nada H. Aldahlawi Arie L. Marcovich Jurriaan Brekelmans Alexandra Goz

Abstract A cross-linking technique involving application of Bacteriochlorophyll Derivative WST-11 mixed with dextran (WST-D) to the epithelium-debrided cornea and illumination with Near Infrared (NIR), has been identified as a promising therapy for stiffening pathologically weakened corneas. To investigate its effect on corneal collagen architecture, x-ray scattering and electron microscopy data were collected from paired WST-D/NIR treated and untreated rabbit corneas. The treated eye received 2.5 mg/mL WST-D and was illuminated by a NIR diode laser (755 nm, 10 mW/cm 2 ). An increase in corneal thickness (caused by corneal oedema) occurred at 1-day post-treatment but resolved in the majority of cases within 4 days. The epithelium was fully healed after 6–8 days. X-ray scattering revealed no difference in average collagen interfibrillar spacing, fibril diameter, D-periodicity or intermolecular spacing between treated and untreated specimens. Similarly, electron microscopy images of the anterior and posterior stroma in healed WST-D/NIR corneas and untreated controls revealed no obvious differences in collagen organisation or fibril diameter. As the size and organisation of stromal collagen is closely associated with the optical properties of the cornea, the absence of any large-scale changes following treatment confirms the potential of WST-D/NIR therapy as a means of safely stiffening the cornea.

Collagen fibril

10.1038/s41598-020-66869-y

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Citations (7)

The Effect of Riboflavin/UVA Collagen Cross-linking Therapy on the Structure and Hydrodynamic Behaviour of the Ungulate and Rabbit Corneal Stroma

PLoS ONE (2013)

Sally Hayes Christina S. Kamma‐Lorger Craig Boote Robert D. Young Andrew J. Quantock

Purpose To examine the effect of riboflavin/UVA corneal crosslinking on stromal ultrastructure and hydrodynamic behaviour. Methods One hundred and seventeen enucleated ungulate eyes (112 pig and 5 sheep) and 3 pairs of rabbit eyes, with corneal epithelium removed, were divided into four treatment groups: Group 1 (28 pig, 2 sheep and 3 rabbits) were untreated; Group 2 (24 pig) were exposed to UVA light (3.04 mW/cm2) for 30 minutes and Group 3 (29 pig) and Group 4 (31 pig, 3 sheep and 3 rabbits) had riboflavin eye drops applied to the corneal surface every 5 minutes for 35 minutes. Five minutes after the initial riboflavin instillation, the corneas in Group 4 experienced a 30 minute exposure to UVA light (3.04 mW/cm2). X-ray scattering was used to obtain measurements of collagen interfibrillar spacing, spatial order, fibril diameter, D-periodicity and intermolecular spacing throughout the whole tissue thickness and as a function of tissue depth in the treated and untreated corneas. The effect of each treatment on the hydrodynamic behaviour of the cornea (its ability to swell in saline solution) and its resistance to enzymatic digestion were assessed using in vitro laboratory techniques. Results Corneal thickness decreased significantly following riboflavin application (p<0.01) and also to a lesser extent after UVA exposure (p<0.05). With the exception of the spatial order factor, which was higher in Group 4 than Group 1 (p<0.01), all other measured collagen parameters were unaltered by cross-linking, even within the most anterior 300 microns of the cornea. The cross-linking treatment had no effect on the hydrodynamic behaviour of the cornea but did cause a significant increase in its resistance to enzymatic digestion. Conclusions It seems likely that cross-links formed during riboflavin/UVA therapy occur predominantly at the collagen fibril surface and in the protein network surrounding the collagen.

Corneal collagen cross-linking

10.1371/journal.pone.0052860

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Citations (168)