A potential mouse model for the erosive vitreoretinopathy of Wagner disease

Patients with the very rare eye pathology Wagner disease (OMIM #143200) present with an abnormal (empty) vitreous, retinal detachment and altered electroretinogram (ERG). The disease is progressive and can eventually lead to blindness. No therapy can be offered to date. The genetic basis is the presence of mutations in the VCAN gene, encoding the large extracellular matrix molecule versican, which is a component of the vitreous. All identified mutations map to the canonical splice sites flanking exon 8, resulting in low number of aberrant splice products and a severe increase in two (V2, V3) of the four naturally occurring splice variants. The pathomechanism of Wagner's disease is poorly understood and a mouse model may afford further insight. The hdf -/- mice, named for their initial phenotype of heart defects, carry a null allele for Vcan that leads to embryonic lethality when homozygous, but heterozygote animals are viable. Here we investigated a possible eye phenotype in the heterozygous animals. While the overall morphology of retina and ciliary body appears to be normal, older (17 months) mutant animals show a decrease in ERG signaling profiles affecting the a-, b- and c-waves. This aspect of altered ERG profile demonstrates similarities to the human disease manifestation and underlines the suitability of heterozygous hdf+/- mice as a model for Wagner disease

Age-related differences in human skin proteoglycans

Previous work has shown that versican, decorin and a catabolic fragment of decorin, termed decorunt, are the most abundant proteoglycans in human skin. Further analysis of versican indicates that four major core protein species are present in human skin at all ages examined from fetal to adult. Two of these are identified as the V0 and V1 isoforms, with the latter predominating. The other two species are catabolic fragments of V0 and V1, which have the amino acid sequence DPEAAE as their carboxyl terminus. Although the core proteins of human skin versican show no major age-related differences, the glycosaminoglycans (GAGs) of adult skin versican are smaller in size and show differences in their sulfation pattern relative to those in fetal skin versican. In contrast to human skin versican, human skin decorin shows minimal age-related differences in its sulfation pattern, although, like versican, the GAGs of adult skin decorin are smaller than those of fetal skin decorin. Analysis of the catabolic fragments of decorin from adult skin reveals the presence of other fragments in addition to decorunt, although the core proteins of these additional decorin catabolic fragments have not been identified. Thus, versican and decorin of human skin show age-related differences, versican primarily in the size and the sulfation pattern of its GAGs and decorin in the size of its GAGs. The catabolic fragments of versican are detected at all ages examined, but appear to be in lower abundance in adult skin compared with fetal skin. In contrast, the catabolic fragments of decorin are present in adult skin, but are virtually absent from fetal skin. Taken together, these data suggest that there are age-related differences in the catabolism of proteoglycans in human skin. These age-related differences in proteoglycan patterns and catabolism may play a role in the age-related changes in the physical properties and injury response of human ski

Determinants of versican-V1 proteoglycan processing by the metalloproteinase ADAMTS5

Proteolysis of the Glu(441)-Ala(442) bond in the glycosaminoglycan (GAG) β domain of the versican-V1 variant by a disintegrin-like and metalloproteinase domain with thrombospondin type 1 motif (ADAMTS) proteases is required for proper embryo morphogenesis. However, the processing mechanism and the possibility of additional ADAMTS-cleaved processing sites are unknown. We demonstrate here that if Glu(441) is mutated, ADAMTS5 cleaves inefficiently at a proximate upstream site but normally does not cleave elsewhere within the GAGβ domain. Chondroitin sulfate (CS) modification of versican is a prerequisite for cleavage at the Glu(441)-Ala(442) site, as demonstrated by reduced processing of CS-deficient or chondroitinase ABC-treated versican-V1. Site-directed mutagenesis identified the N-terminal CS attachment sites Ser(507) and Ser(525) as essential for processing of the Glu(441)-Ala(442) bond by ADAMTS5. A construct including only these two GAG chains, but not downstream GAG attachment sites, was cleaved efficiently. Therefore, CS chain attachment to Ser(507) and Ser(525) is necessary and sufficient for versican proteolysis by ADAMTS5. Mutagenesis of Glu(441) and an antibody to a peptide spanning Thr(432)-Gly(445) (i.e. containing the scissile bond) reduced versican-V1 processing. ADAMTS5 lacking the C-terminal ancillary domain did not cleave versican, and an ADAMTS5 ancillary domain construct bound versican-V1 via the CS chains. We conclude that docking of ADAMTS5 with two N-terminal GAG chains of versican-V1 via its ancillary domain is required for versican processing at Glu(441)-Ala(442). V1 proteolysis by ADAMTS1 demonstrated a similar requirement for the N-terminal GAG chains and Glu(441). Therefore, versican cleavage can be inhibited substantially by mutation of Glu(441), Ser(507), and Ser(525) or by an antibody to the region of the scissile bond

Inter-laboratory validation of PCR-based HPV detection in pathology specimens

The detection and typing of human papilloma virus (HPV) in pathology specimens is gaining increasingly in importance. In the context of the initiative for quality assurance in pathology (QuIP) of the German Society of Pathology and the Professional Association of German Pathologists, four panel laboratories with experience and expertise in polymerase chain reaction (PCR)-based HPV detection were selected to establish an inter-laboratory trial. In a first step, these laboratories performed an internal testing of their own methodologies, which comprised DNA sequencing, multiplex nested PCR and hybridization techniques. Material from 39 samples including paraffin sections and DNA preparations of tissues and plasmids were evaluated by each panel institute according to their own protocols. Despite the different methodologies, a high degree of inter-laboratory reliability was achieved. In this report, we summarise the results. Pretested specimens are available for the external trail and can be ordered from the steering institute via provitro GmbH Berlin ( http://www.provitro.de ). Supplementary data are online available at http://pathologie-ccm.charite.de (rubric "Forschung"), which includes a web-based photo gallery of HPV-associated lesions and their potential association with specific virus types. The initiative is intended to foster the quality assurance of molecular HPV analysis in pathology and its correlation with morphological changes

Imbalanced Expression of <i>Vcan</i> mRNA Splice Form Proteins Alters Heart Morphology and Cellular Protein Profiles

<div><p>The fundamental importance of the proteoglycan versican to early heart formation was clearly demonstrated by the <i>Vcan</i> null mouse called heart defect (<i>hdf</i>). Total absence of the <i>Vcan</i> gene halts heart development at a stage prior to the heart’s pulmonary/aortic outlet segment growth. This creates a problem for determining the significance of versican’s expression in the forming valve precursors and vascular wall of the pulmonary and aortic roots. This study presents data from a mouse model, <i>Vcan</i>^(tm1Zim), of heart defects that results from deletion of exon 7 in the <i>Vcan</i> gene. Loss of exon 7 prevents expression of two of the four alternative splice forms of the <i>Vcan</i> gene. Mice homozygous for the exon 7 deletion survive into adulthood, however, the inability to express the V2 or V0 forms of versican results in ventricular septal defects, smaller cushions/valve leaflets with diminished myocardialization and altered pulmonary and aortic outflow tracts. We correlate these phenotypic findings with a large-scale differential protein expression profiling to identify compensatory alterations in cardiac protein expression at E13.5 post coitus that result from the absence of <i>Vcan</i> exon 7. The <i>Vcan</i>^(tm1Zim) hearts show significant changes in the relative abundance of several cytoskeletal and muscle contraction proteins including some previously associated with heart disease. These alterations define a protein fingerprint that provides insight to the observed deficiencies in pre-valvular/septal cushion mesenchyme and the stability of the myocardial phenotype required for alignment of the outflow tract with the heart ventricles.</p></div

Splice forms of versican.

<p>The <i>Vcan</i> gene consists of 15 exons. Four mRNA and corresponding protein variants of the core protein (V0, V1, V2, V3) are derived from alternative splicing of exons 7 (blue boxes) and/or 8 (green boxes) into mRNA of <i>Vcan</i> gene. The glycosaminoglycan attachment domains GAG α & β are encoded by exon 7 (blue) and 8 (green) respectively. Deletion of exon 7 (blue) results in the loss of both the V2 and V0 variants. Exons 2–6 comprise the G1 domain that binds hyaluronan and 9–15 the G3 domain that also can interact with other ECM molecules.</p

Western blot comparison of four proteins with altered abundance by itraq in <i>Vcan</i>^(tm1Zim) mutant and wild-type hearts.

<p>Proteins that showed different levels of altered abundance in the <i>Vcan</i>^(tm1Zim) hearts are shown in the blot. Annexin A6 and Stathmin are proteins expressed in the heart that showed a relative decrease (0.85x and 0.73x respectively) in abundance in the <i>Vcan</i>^(tm1Zim) mutant by iTRAQ and serphin1 (Hsp 47) that by iTRAQ showed increased abundance (1.55x). Additionally, Desmin that did not change with any significance between mutant (Mut) and wild-type (Wt) by iTRAQ, showed no significant change by western blot. Results of average relative density measurements of separately analyzed hearts are shown in the graph for each protein (for Stmn, Hsp47, Desmin n = 3; p<0.01; for Annexin A6 n = 2; p<0.0045).</p

Three-dimensional reconstructions and quantitative measurements of wild-type and <i>Vcan</i> exon 7 AV associated cushions in E13.5 pc hearts.

<p>The differences found in the mesenchymalized AV cushions, visually apparent in the 3-dimensional comparisons (Panel A, ventral view; B, dorsal view), were also quantified using the AMIRA imaging software to measure the cushion volumes. A significant reduction in volume was measured in the central AV cushion comprising the aortic leaflets (AL in panels A, B; reduced 35%; *p<0.034 n = 3 for each genotype, panel C) and septal (SL in panels A,B; reduced 30% p<0.046 n = 3 for each genotype in panel D). A significant (*p value 0.05 panel G) decrease in volume (0.58x) of the dorsal mesenchymal protrusion (DM) was measured. The other cushions showed no significant difference and the overall size of the E13.5 <i>Vcan</i>^(tm1Zim) and wild-type hearts (measured by tissue weight) was not significantly different (74 mg and 75.6 mg respectively; n = 3 for each genotype). AL-aortic leaflet (red); PL-parietal leaflet (blue); SL-septal leaflet (pink); ML-mural leaflet (green); DM-dorsal mesenchyme (white).</p

Three-dimensional reconstruction and quantitative comparisons of wild-type and <i>Vcan</i> exon 7 cardiac OT associated cushions in E13.5 pc hearts.

<p>Significant differences were found in the volume of the pulmonary lumen and size of the pulmonary cushions in the <i>Vcan</i>^(tm1Zim) compared to wild-type littermates. The differences, visually apparent in the 3-dimensional comparisons (panel A; wild-type and <i>Vcan</i>^(tm1Zim)), were quantified using the AMIRA imaging software to measure the volumes of the cushion primordia of the pulmonary and aortic valves and of the pulmonary (brown) and aortic (purple) lumens. A significant increase (51%; *P value 0.047 panel D) was found in the size of the pulmonary cushions in the <i>Vcan</i>^(tm1Zim) hearts compared to wild-type littermates (panels D). The pulmonary lumens of the <i>Vcan</i>^(tm1Zim) cardiac outlets were found to be significantly (panel E; *P value 0.028 ) smaller (57%) then that of wild-type littermate controls. The aortic lumen volumes measured smaller but did not show a significant change (panel C). Brown-pulmonary lumen (PL); purple-aortic lumen (Ao); green-pulmonary cushions; yellow-aortic cushions.</p