Novel deletions causing pseudoxanthoma elasticum underscore the genomic instability of the ABCC6 region

Mutations in ABCC6 cause pseudoxanthoma elasticum (PXE), a heritable disease that affects elastic fibers. Thus far, >200 mutations have been characterized by various PCR-based techniques (primarily direct sequencing), identifying up to 90% of PXE-causing alleles. This study wanted to assess the importance of deletions and insertions in the ABCC6 genomic region, which is known to have a high recombinational potential. To detect ABCC6 deletions/insertions, which can be missed by direct sequencing, multiplex ligation-dependent probe amplification (MLPA) was applied in PXE patients with an incomplete genotype. MLPA was performed in 35 PXE patients with at least one unidentified mutant allele after exonic sequencing and exclusion of the recurrent exon 23-29 deletion. Six multi-exon deletions and four single-exon deletions were detected. Using MLPA in addition to sequencing, we expanded the ABCC6 mutation spectrum with 9 novel deletions and characterized 25% of unidentified disease alleles. Our results further illustrate the instability of the ABCC6 genomic region and stress the importance of screening for deletions in the molecular diagnosis of PXE. Journal of Human Genetics (2010) 55, 112-117; doi: 10.1038/jhg.2009.132; published online 15 January 201

Hyaluronan uptake by adult human skin fibroblasts in vitro

Low and high molecular weight hyaluronan (HA) was added to adult human fibroblasts grown in monolayer to assess its influence on CD44 expression, its internalisation and effect on cell growth. CD44 expression on the surface of in vitro fibroblasts was not modified by different concentrations of FCS, whereas it was sensitive to cell cycle, being higher in the growing than in the resting phase. Independently from molecular weight, upon addition of exogenous HA (from 0.1 up to 1 mg/mL) to fibroblasts in the growing phase, a slight but constant decrease of the expression of CD44 on the surface of fibroblasts was observed; moreover, HA induced a rearrangement of CD44 into patches in close relationship with the terminal regions of stress fibers, which became thicker and more rigid after a few hours from the addition of HA to the medium. Fluorescent HA, added to the culture medium, rapidly attached to the plasma membrane and in less than two minutes was observed within cells, partly in association with its receptor CD44. By the contemporary use of neutral red, which accumulates into functional lysosomes, the great majority of internalised HA was found within lysosomes. HA receptor RHAMM-IHABP was rather homogeneously localised within the cytoplasm of normal growing fibroblasts. Upon addition of HA, the RHAMM-IHABP distribution became discontinuous around the nucleus. Addition of HA to fibroblasts induced a significant inhibition of cell growth, which was dependent on HA concentration and irrespective of HA molecular weight, at least in the ranges tested. Results show that extra-cellular HA is rapidly taken up by human dermal fibroblasts together with its CD44 receptor, and transported mostly to the lysosomes. Both low and high molecular weight HA induced down-regulation of cell proliferation, which would seem to be mediated by HA catabolism

Effects of Hylan G-F 20 supplementation on cartilage preservation detected by magnetic resonance imaging in osteoarthritis of the knee: a two-year single-blind clinical trial

<p>Abstract</p> <p>Background</p> <p>Although viscosupplementation is an effective symptomatic treatment for knee osteoarthritis (OA), the effect of longer term administration on articular cartilage has not been fully explored. We examined the effect of viscosupplementation with Hylan G-F 20 on knee cartilage over 2 years in patients with knee OA.</p> <p>Methods</p> <p>In this prospective, single-blind, parallel control group pilot study, 78 patients with symptomatic knee OA (Kellgren-Lawrence grade II and III) were assigned to either intervention group (n = 39 receiving 4 courses of 3 × 2.0 ml of intra-articular Hylan G-F 20 injections at 6 month intervals) or control group (n = 39 receiving usual care for knee OA without injections). Magnetic resonance imaging of the study knee was performed at baseline, 12 and 24 months. Cartilage volume and defects were assessed using validated methods.</p> <p>Results</p> <p>Fifty-five subjects (71%) completed 24 month follow up. Over 24 months, the intervention group had a reduced annual percentage rate of medial and lateral tibial cartilage volume loss (mean ± SD, -0.3 ± 2.7% and -1.4 ± 4.3%) compared with the control group (2.3 ± 2.6% and 1.4 ± 2.6%, P = 0.001 and 0.005 for difference, respectively). The intervention group also showed reduced cartilage defect score increment in the medial tibiofemoral compartment (0.1 ± 1.3) compared with the control group (0.8 ± 1.5, P = 0.05).</p> <p>Conclusions</p> <p>Six monthly intra-articular injections of Hylan G-F 20 administered to patients with symptomatic knee OA have a beneficial effect on knee cartilage preservation measured by both cartilage volume and cartilage defects. Hylan G-F 20 warrants further evaluation in larger clinical trials as a possible disease-modifying agent in the treatment of knee OA.</p> <p>Trial Registration</p> <p>The study was registered with ClinicalTrials.gov (<a href="http://www.clinicaltrials.gov/ct2/show/NCT00393393">NCT00393393</a>).</p

Identification of Histological Patterns in Clinically Affected and Unaffected Palm Regions in Dupuytren's Disease

Dupuytren's disease is a fibro-proliferative disease characterized by a disorder of the extracellular matrix (ECM) and high myofibroblast proliferation. However, studies failed to determine if the whole palm fascia is affected by the disease. The objective of this study was to analyze several components of the extracellular matrix of three types of tissues—Dupuytren's diseased contracture cords (DDC), palmar fascia clinically unaffected by Dupuytren's disease contracture (NPF), and normal forehand fascia (NFF). Histological analysis, quantification of cells recultured from each type of tissue, mRNA microarrays and immunohistochemistry for smooth muscle actin (SMA), fibrillar ECM components and non-fibrillar ECM components were carried out. The results showed that DDC samples had abundant fibrosis with reticular fibers and few elastic fibers, high cell proliferation and myofibroblasts, laminin and glycoproteins, whereas NFF did not show any of these findings. Interestingly, NPF tissues had more cells showing myofibroblasts differentiation and more collagen and reticular fibers, laminin and glycoproteins than NFF, although at lower level than DDC, with similar elastic fibers than DDC. Immunohistochemical expression of decorin was high in DDC, whereas versican was highly expressed NFF, with no differences for aggrecan. Cluster analysis revealed that the global expression profile of NPF was very similar to DDC, and reculturing methods showed that cells corresponding to DDC tissues proliferated more actively than NPF, and NPF more actively than NFF. All these results suggest that NPF tissues may be affected, and that a modification of the therapeutic approach used for the treatment of Dupuytren's disease should be considered.This work was supported by CTS-115 (Tissue Engineering Group), University of Granada/Spain

Vitamin K supplementation increases vitamin K tissue levels but fails to counteract ectopic calcification in a mouse model for pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE) is an autosomal recessive disorder in which calcification of connective tissue leads to pathology in skin, eye and blood vessels. PXE is caused by mutations in ABCC6. High expression of this transporter in the basolateral hepatocyte membrane suggests that it secretes an as-yet elusive factor into the circulation which prevents ectopic calcification. Utilizing our Abcc6−/− mouse model for PXE, we tested the hypothesis that this factor is vitamin K (precursor) (Borst et al. 2008, Cell Cycle). For 3 months, Abcc6−/− and wild-type mice were put on diets containing either the minimum dose of vitamin K required for normal blood coagulation or a dose that was 100 times higher. Vitamin K was supplied as menaquinone-7 (MK-7). Ectopic calcification was monitored in vivo by monthly micro-CT scans of the snout, as the PXE mouse model develops a characteristic connective tissue mineralization at the base of the whiskers. In addition, calcification of kidney arteries was measured by histology. Results show that supplemental MK-7 had no effect on ectopic calcification in Abcc6−/− mice. MK-7 supplementation increased vitamin K levels (in skin, heart and brain) in wild-type and in Abcc6−/− mice. Vitamin K tissue levels did not depend on Abcc6 genotype. In conclusion, dietary MK-7 supplementation increased vitamin K tissue levels in the PXE mouse model but failed to counteract ectopic calcification. Hence, we obtained no support for the hypothesis that Abcc6 transports vitamin K and that PXE can be cured by increasing tissue levels of vitamin K

Elastogenic Protein Expression of a Highly Elastic Murine Spinal Ligament: The Ligamentum Flavum

Spinal ligaments, such as the ligamentum flavum (LF), are prone to degeneration and iatrogenic injury that can lead to back pain and nerve dysfunction. Repair and regeneration strategies for these tissues are lacking, perhaps due to limited understanding of spinal ligament formation, the elaboration of its elastic fibers, maturation and homeostasis. Using immunohistochemistry and histology, we investigated murine LF elastogenesis and tissue formation from embryonic to mature postnatal stages. We characterized the spatiotemporal distribution of the key elastogenic proteins tropoelastin, fibrillin-1, fibulin-4 and lysyl oxidase. We found that elastogenesis begins in utero with the microfibril constituent fibrillin-1 staining intensely just before birth. Elastic fibers were first detected histologically at postnatal day (P) 7, the earliest stage at which tropoelastin and fibulin-4 stained intensely. From P7 to P28, elastic fibers grew in diameter and became straighter along the axis. The growth of elastic fibers coincided with intense staining of tropoelastin and fibulin-4 staining, possibly supporting a chaperone role for fibulin-4. These expression patterns correlated with reported skeletal and behavioral changes during murine development. This immunohistochemical characterization of elastogenesis of the LF will be useful for future studies investigating mechanisms for elastogenesis and developing new strategies for treatment or regeneration of spinal ligaments and other highly elastic tissues

Wound dressings for a proteolytic-rich environment

Wound dressings have experienced continuous and significant changes over the years based on the knowledge of the biochemical events associated with chronic wounds. The development goes from natural materials used to just cover and conceal the wound to interactive materials that can facilitate the healing process, addressing specific issues in non-healing wounds. These new types of dressings often relate with the proteolytic wound environment and the bacteria load to enhance the healing. Recently, the wound dressing research is focusing on the replacement of synthetic polymers by natural protein materials to delivery bioactive agents to the wounds. This article provides an overview on the novel protein-based wound dressings such as silk fibroin keratin and elastin. The improved properties of these dressings, like the release of antibiotics and growth factors, are discussed. The different types of wounds and the effective parameters of healing process will be reviewed