Paul N. Bishop

Structural macromolecules and supramolecular organisation of the vitreous gel

The vitreous gel is a transparent extracellular matrix that fills the cavity behind the lens of the eye and is surrounded by and attached to the retina. This gel liquefies during ageing and in 25-30% of the oppulation the residual gel structure eventually collapses away from the posterior retina in a process called posterior retina in a process called posterior vitreous detachment. This process plays a pivotal role in a number of common blinding conditions including rhegmatogenous retinal detachment, proliferative diabetic retinopathy and macular hole formation. In order to understand the molecular events underlying vitreous liquefaction and posterior vitreous detachment and to develop new therapies it is important to understand the molecular basis of normal vitreous gel structure and how this is altered during ageing. It has previously been established that a dilute dispersion of thin (heterotypic) collagen fibrils is essential to the gel structure and that age-related vitreous liquefaction is intimately related to a process whereby these collagen fibrils aggregate. Collagen fibrils have a natural tendency to aggregate so a key question that has to be addressed is: what normally maintains the spacing of the collagen fibrils? In mammalian vitreous a network of hyaluronan normally fills the spaces between these collagen fibrils. This hyaluronan network can be removed without destroying the gel structure, so the hyaluronan is not essential for maintaining the spacing of the collagen fibrils although it probably does increase the mechanical resilience of the gel. The thin heterotypic collagen fibrils have a coating of non-covalently bound macromolecules which, along with the surface features of the collagen fibrils themselves, probably play a fundamental role in maintaining gel stability. They are likely to both maintain the short-range spacing of vitreous collagen fibrils and to link the fibrils together to form a contiguous network. A collagen fibril-associated macromolecule that may contribute to the maintenance of short-range spacing is opticin, a newly discovered extracellular matrix leucine-rich repeat protein. In addition, surface features of the collagen fibrils such as the chondroitin sulphate glycosaminoglycan chains of type IX collagen proteoglycan may also play an important role in maintaining fibril spacing. Furthering our knowledge of these and other components related to the surface of the heterotypic collagen fibrils will allow us to make important strides in understanding the macromolecular organisation of this unique and fascinating tissue. In addition, it will open up new therapeutic opportunities as it will allow the development of therapeutic reagents that can be used to modulate vitreous gel structure and thus treat a number of common, potentially blinding, ocular conditions.

Comprehensive Validation of Cardiovascular Magnetic Resonance Techniques for the Assessment of Myocardial Extracellular Volume

Background— Extracellular matrix expansion is a key element of ventricular remodeling and a potential therapeutic target. Cardiovascular magnetic resonance (CMR) T1-mapping techniques are increasingly used to evaluate myocardial extracellular volume (ECV); however, the most widely applied methods are without histological validation. Our aim was to perform comprehensive validation of (1) dynamic-equilibrium CMR (DynEq-CMR), where ECV is quantified using hematocrit-adjusted myocardial and blood T1 values measured before and after gadolinium bolus; and (2) isolated measurement of myocardial T1, used as an ECV surrogate. Methods and Results— Whole-heart histological validation was performed using 96 tissue samples, analyzed for picrosirius red collagen volume fraction, obtained from each of 16 segments of the explanted hearts of 6 patients undergoing heart transplantation who had prospectively undergone CMR before transplantation (median interval between CMR and transplantation, 29 days). DynEq-CMR–derived ECV was calculated from T1 measurements made using a modified Look-Locker inversion recovery sequence before and 10 and 15 minutes post contrast. In addition, ECV was measured 2 to 20 minutes post contrast in 30 healthy volunteers. There was a strong linear relationship between DynEq-CMR–derived ECV and histological collagen volume fraction (P<0.001; within-subject: r=0.745; P<0.001; r 2=0.555 and between-subject: r=0.945; P<0.01; r 2=0.893; for ECV calculated using 15-minute postcontrast T1). Correlation was maintained throughout the entire heart. Isolated postcontrast T1 measurement showed significant within-subject correlation with histological collagen volume fraction (r=−0.741; P<0.001; r 2=0.550 for 15-minute postcontrast T1), but between-subject correlations were not significant. DynEq-CMR–derived ECV varied significantly according to contrast dose, myocardial region, and sex. Conclusions— DynEq-CMR–derived ECV shows a good correlation with histological collagen volume fraction throughout the whole heart. Isolated postcontrast T1 measurement is insufficient for ECV assessment.

Adult vitreous structure and postnatal changes

This review will focus on the molecular organisation of the adult vitreous and how it undergoes ageing changes throughout life that result in vitreous liquefaction and a predisposition towards posterior vitreous detachment and retinal break formation. At birth, the vitreous humour is in a gel state due to the presence of a network of fine collagen fibrils. With ageing, these collagen fibrils progressively aggregate due to a loss of type IX collagen from their surfaces. The aggregation of collagen fibrils may cause vitreous liquefaction which, when combined with an age-related weakening of postbasal vitreoretinal adhesion, predisposes to posterior vitreous detachment. Throughout postnatal life, the posterior border of the vitreous base migrates posteriorly from the ora serrata into the peripheral retina. This is due to new collagen synthesis by the peripheral retina. This new collagen intertwines with pre-existing cortical vitreous collagen to create new adhesions and thereby extends the vitreous base posteriorly. If irregularities in the posterior border of the vitreous base arise from this process, there is a predisposition towards retinal break formation during posterior vitreous detachment and subsequent rhegmatogenous retinal detachment.

Impaired Binding of the Age-related Macular Degeneration-associated Complement Factor H 402H Allotype to Bruch's Membrane in Human Retina

Age-related macular degeneration (AMD) is the predominant cause of blindness in the industrialized world where destruction of the macula, i.e. the central region of the retina, results in loss of vision. AMD is preceded by the formation of deposits in the macula, which accumulate between the Bruchs membrane and the retinal pigment epithelium (RPE). These deposits are associated with complement-mediated inflammation and perturb retinal function. Recent genetic association studies have demonstrated that a common allele (402H) of the complement factor H (CFH) gene is a major risk factor for the development of AMD; CFH suppresses complement activation on host tissues where it is believed to bind via its interaction with polyanionic structures. We have shown previously that this coding change (Y402H; from a tyrosine to histidine residue) alters the binding of the CFH protein to sulfated polysaccharides. Here we demonstrate that the AMD-associated polymorphism profoundly affects CFH binding to sites within human macula. Notably, the AMD-associated 402H variant binds less well to heparan sulfate and dermatan sulfate glycosaminoglycans within Bruchs membrane when compared with the 402Y form; both allotypes exhibit a similar level of binding to the RPE. We propose that the impaired binding of the 402H variant to Bruchs membrane results in an overactivation of the complement pathway leading to local chronic inflammation and thus contributes directly to the development and/or progression of AMD. These studies therefore provide a putative disease mechanism and add weight to the genetic association studies that implicate the 402H allele as an important risk factor in AMD.

Tissue-Specific Host Recognition by Complement Factor H Is Mediated by Differential Activities of Its Glycosaminoglycan-Binding Regions

Complement factor H (CFH) regulates complement activation in host tissues through its recognition of polyanions, which mediate CFH binding to host cell surfaces and extracellular matrix, promoting the deactivation of deposited C3b. These polyanions include heparan sulfate (HS), a glycosaminoglycan with a highly diverse range of structures, for which two regions of CFH (CCP6–8 and CCP19–20) have been implicated in HS binding. Mutations/polymorphisms within these glycosaminoglycan-binding sites have been associated with age-related macular degeneration (AMD) and atypical hemolytic uremic syndrome. In this study, we demonstrate that CFH has tissue-specific binding properties mediated through its two HS-binding regions. Our data show that the CCP6–8 region of CFH binds more strongly to heparin (a highly sulfated form of HS) than CCP19–20, and that their sulfate specificities are different. Furthermore, the HS binding site in CCP6–8, which is affected by the AMD-associated Y402H polymorphism, plays the principal role in host tissue recognition in the human eye, whereas the CCP19–20 region makes the major contribution to the binding of CFH in the human kidney. This helps provide a biochemical explanation for the genetic basis of tissue-specific diseases such as AMD and atypical hemolytic uremic syndrome, and leads to a better understanding of the pathogenic mechanisms for these diseases of complement dysregulation.

Matrix Metalloproteinase-1 Associates with Intracellular Organelles and Confers Resistance to Lamin A/C Degradation during Apoptosis

Since the first description of matrix metalloproteinase (MMP)-1 as an interstitial collagenase, great importance has been ascribed to this enzyme in extracellular matrix remodeling during tumoral, inflammatory, and angiogenic processes. As more evidence for the role of MMPs in targeting nonmatrix substrates emerges, casual observations that intracellular MMP-1 is found in vitro and in vivo prompt investigation of the role that MMP-1 may play on basic cell functions such as cell division and apoptosis. Here we show for the first time that MMP-1 not only has extracellular functions but that it is strongly associated with mitochondria and nuclei and accumulates within the cells during the mitotic phase of the cell cycle. On induction of apoptosis, MMP-1 co-localized with aggregated mitochondria and accumulated around fragmented nuclei. Inhibition of this enzyme by RNA interference or treatment with a broad MMP inhibitor caused faster degradation of lamin A, activation of caspases, and fragmentation of DNA when compared with untreated cells. These observations strongly suggest that intracellular association of MMP-1 to mitochondria and nuclei confers resistance to apoptosis and may explain the well-known association of this enzyme with tumor cell survival and spreading.

Identification in Vitreous and Molecular Cloning of Opticin, a Novel Member of the Family of Leucine-rich Repeat Proteins of the Extracellular Matrix

A prominent 45-kDa component was identified by protein staining following SDS-polyacrylamide gel electrophoresis of a 4 m guanidine hydrochloride extract from bovine vitreous collagen fibrils. Peptide sequences obtained from this component were used as a basis for the cloning (from human retinal cDNA) and sequencing of a novel member of the leucine-rich repeat extracellular matrix protein family that we have named opticin. Opticin mRNA was found by reverse transcription polymerase chain reaction in ligament and skin as well as in retina. An open reading frame containing 332 amino acids was identified, the first 19 amino acids representing a signal peptide. The deduced amino acid sequence of the mature protein encodes a 35-kDa protein with a calculated isoelectric point of 5.4. The central domain of this protein consists of six B-type leucine-rich repeats. This domain is flanked by cysteine clusters including a C-terminal two-cysteine cluster containing an additional leucine-rich repeat. The N-terminal region contains a cluster of potential O-glycosylation sites, and analysis of bovine vitreous opticin demonstrated the presence of sialylatedO-linked oligosaccharides substituting the core protein. Opticin shows highest protein sequence identity to epiphycan (42%) and osteoglycin (35%) and belongs to Class III of the leucine-rich repeat extracellular matrix protein family.

The role of the posterior ciliary body in the biosynthesis of vitreous humour.

Recently, several groups have published new information regarding the origins and structure of the vitreous humour, and the inner limiting lamina (ILL) of the retina. This short article provides an overview of this new information. It is proposed that vitreous proteins are derived from several different cell types with the posterior half of the non-pigmented ciliary epithelium being prominent in the expression of several connective tissue macromolecules. In addition, some basement membrane macromolecules are also expressed by the ciliary body and may subsequently be assembled on the surface of the Müller cells to form the ILL. New data suggest that the posterior half of the non-pigmented ciliary epithelium has substantial secretory activity and is likely to play a pivotal role in eye development.

Crystal Structure of the Biglycan Dimer and Evidence That Dimerization Is Essential for Folding and Stability of Class I Small Leucine-rich Repeat Proteoglycans

Biglycan and decorin are two closely related proteoglycans whose protein cores contain leucine-rich repeats flanked by disulfides. We have previously shown that decorin is dimeric both in solution and in crystal structures. In this study we determined whether biglycan dimerizes and investigated the role of dimerization in the folding and stability of these proteoglycans. We used light scattering to show that biglycan is dimeric in solution and solved the crystal structure of the glycoprotein core of biglycan at 3.40-Å resolution. This structure reveals that biglycan dimerizes in the same way as decorin, i.e. by apposition of the concave inner surfaces of the leucine-rich repeat domains. We demonstrate that low concentrations of guanidinium chloride denature biglycan and decorin but that the denaturation is completely reversible following removal of the guanidinium chloride, as assessed by circular dichroism spectroscopy. Furthermore, the rate of refolding is dependent on protein concentration, demonstrating that it is not a unimolecular process. Upon heating, decorin shows a single structural transition at a Tm of 45-46 °C but refolds completely upon cooling to 25 °C. This property of decorin enabled us to show both by calorimetry and light scattering that dimer to monomer transition coincided with unfolding and monomer to dimer transition coincided with refolding; thus these processes are inextricably linked. We further conclude that folded monomeric biglycan or decorin cannot exist in solution. This implies novel interrelated functions for the parallel β sheet faces of these leucine-rich repeat proteoglycans, including dimerization and stabilization of protein folding.

Genome-wide association study of age-related macular degeneration identifies associated variants in the TNXB–FKBPL–NOTCH4 region of chromosome 6p21.3

Age-related macular degeneration (AMD) is a leading cause of visual loss in Western populations. Susceptibility is influenced by age, environmental and genetic factors. Known genetic risk loci do not account for all the heritability. We therefore carried out a genome-wide association study of AMD in the UK population with 893 cases of advanced AMD and 2199 controls. This showed an association with the well-established AMD risk loci ARMS2 (age-related maculopathy susceptibility 2)-HTRA1 (HtrA serine peptidase 1) (P =2.7 × 10(-72)), CFH (complement factor H) (P =2.3 × 10(-47)), C2 (complement component 2)-CFB (complement factor B) (P =5.2 × 10(-9)), C3 (complement component 3) (P =2.2 × 10(-3)) and CFI (P =3.6 × 10(-3)) and with more recently reported risk loci at VEGFA (P =1.2 × 10(-3)) and LIPC (hepatic lipase) (P =0.04). Using a replication sample of 1411 advanced AMD cases and 1431 examined controls, we confirmed a novel association between AMD and single-nucleotide polymorphisms on chromosome 6p21.3 at TNXB (tenascin XB)-FKBPL (FK506 binding protein like) [rs12153855/rs9391734; discovery P =4.3 × 10(-7), replication P =3.0 × 10(-4), combined P =1.3 × 10(-9), odds ratio (OR) = 1.4, 95% confidence interval (CI) = 1.3-1.6] and the neighbouring gene NOTCH4 (Notch 4) (rs2071277; discovery P =3.2 × 10(-8), replication P =3.8 × 10(-5), combined P =2.0 × 10(-11), OR = 1.3, 95% CI = 1.2-1.4). These associations remained significant in conditional analyses which included the adjacent C2-CFB locus. TNXB, FKBPL and NOTCH4 are all plausible AMD susceptibility genes, but further research will be needed to identify the causal variants and determine whether any of these genes are involved in the pathogenesis of AMD.