Bipolar anastomosis technique with removable instruments: an easy, fast, and reliable technique for vascular anastomosis

The interrupted suture technique is most commonly used for microsurgical vascular anastomosis. For several reasons (e.g., exposure of suture material to blood, time needed), many attempts have been made to find other solutions. This article describes a new means of performing a microsurgical vascular anastomosis. The aim of this study was to show the feasibility and possible advantages of this new technique. The basic components at work here are a modified cuff and electrically generated heat used to unite the vessel walls. In this way, both endothelial layers are adapted without manipulating the inside of the vessel or leaving behind foreign matter. Various energy/coagulation time settings were used to perform arterial anastomoses (n = 42) in an isogeneic abdominal aorta interposition model in the rat. The quality of anastomosis was evaluated at days 1, 10, 21, and 120. Immediately after the welding process all anastomoses (n = 42) were patent. No stenosis was found at any observation time. Anastomosis time ranged from 3 to 18 minutes (average, 11 minutes). This new technique permits a vascular anastomosis to be performed easily and reliably with a high patency rate. With this technique, the authors are convinced that a skilled surgeon can create a high-quality anastomosis in a fraction of the time needed to sew an anastomosis.     

Complexes of matrilin-1 and biglycan or decorin connect collagen VI microfibrils to both collagen II and aggrecan

Native supramolecular assemblies containing collagen VI microfibrils and associated extracellular matrix proteins were isolated from Swarm rat chondrosarcoma tissue. Their composition and spatial organization were characterized by electron microscopy and immunological detection of molecular constituents. The small leucine-rich repeat (LRR) proteoglycans biglycan and decorin were bound to the N-terminal region of collagen VI. Chondroadherin, another member of the LRR family, was identified both at the N and C termini of collagen VI. Matrilin-1, -3, and -4 were found in complexes with biglycan or decorin at the N terminus. The interactions between collagen VI, biglycan, decorin, and matrilin-1 were studied in detail and revealed a biglycan/matrilin-1 or decorin/matrilin-1 complex acting as a linkage between collagen VI microfibrils and aggrecan or alternatively collagen II. The complexes between matrilin-1 and biglycan or decorin were also reconstituted in vitro. Colocalization of collagen VI and the different ligands in the pericellular matrix of cultured chondrosarcoma cells supported the physiological relevance of the observed interactions in matrix assembly.     

Mosaic chromosomal aberrations in synovial fibroblasts of patients with rheumatoid arthritis, osteoarthritis, and other inflammatory joint diseases

Chromosomal aberrations were comparatively assessed in nuclei extracted from synovial tissue, primary-culture (P-0) synovial cells, and early-passage synovial fibroblasts (SFB; 98% enrichment; P-1, P-4 [passage 1, passage 4]) from patients with rheumatoid arthritis (RA; n = 21), osteoarthritis (OA; n = 24), and other rheumatic diseases. Peripheral blood lymphocytes (PBL) and skin fibroblasts (FB) (P-1, P-4) from the same patients, as well as SFB from normal joints and patients with joint trauma (JT) (n = 4), were used as controls. Analyses proceeded by standard GTG-banding and interphase centromere fluorescence in situ hybridization. Structural chromosomal aberrations were observed in SFB (P-1 or P-4) from 4 of 21 RA patients (19%), with involvement of chromosome 1 [e.g. del(1)(q12)] in 3 of 4 cases. In 10 of the 21 RA cases (48%), polysomy 7 was observed in P-1 SFB. In addition, aneusomies of chromosomes 4, 6, 8, 9, 12, 18, and Y were present. The percentage of polysomies was increased in P-4. Similar chromosomal aberrations were detected in SFB of OA and spondylarthropathy patients. No aberrations were detected in i) PBL or skin FB from the same patients (except for one OA patient with a karyotype 45,X[10]/46,XX[17] in PBL and variable polysomies in long-term culture skin FB); or ii) synovial tissue and/or P-1 SFB of normal joints or of patients with joint trauma. In conclusion, qualitatively comparable chromosomal aberrations were observed in synovial tissue and early-passage SFB of patients with RA, OA, and other inflammatory joint diseases. Thus, although of possible functional relevance for the pathologic role of SFB in RA, these alterations probably reflect a common response to chronic inflammatory stress in rheumatic diseases.     

Translocation mechanism of long sugar chains across the maltoporin membrane channel

Maltoporin allows permeation of long maltodextrin chains. It tightly binds the amphiphilic sugar, offering both hydrophobic interactions with a helical lane of aromatic residues and H bonds with ionic side chains. The minimum-energy path of maltohexaose translocation is obtained by the conjugate peak refinement method, which optimizes a continuous string of conformers without applying constraints. This reveals that the protein is passive while the sugar glides screw-like along the aromatic lane. Near instant switching of sugar hydroxyl H bond partners results in two small energy barriers (of approximately 4 kcal/mol each) during register shift by one glucosyl unit, in agreement with a kinetic analysis of experimental dissociation rates for varying sugar chain lengths. Thus, maltoporin functions like an efficient translocation "enzyme," and the slow rate of the register shift (approximately 1/ms) is due to high collisional friction.     

Matrilin-4 is processed by ADAMTS-5 in late Golgi vesicles present in growth plate chondrocytes of defined differentiation state

The two aggrecanases ADAMTS-4 and ADAMTS-5 have been shown to not only play roles in the breakdown of cartilage extracellular matrix in osteoarthritis, but also mediate processing of matrilins in the secretory pathway. The matrilins are adaptor proteins with a function in connecting fibrillar and network-like components in the cartilage extracellular matrix. Cleavage resulting in processed matrilins with fewer ligand-binding subunits could make these less efficient in providing matrix cohesion. In this study, the processing and degradation of matrilin-4 during cartilage remodeling in the growth plate of the developing mouse long bones were studied in greater detail. We show that ADAMTS-5 and a matrilin-4 neoepitope, revealed upon ADAMTS cleavage, colocalize in prehypertrophic/hypertrophic chondrocytes while they are not detected in proliferating chondrocytes of the growth plate. ADAMTS-5 and the cleaved matrilin-4 are preferentially detected in vesicles derived from the Golgi apparatus. The matrilin-4 neoepitope was not observed in the growth plate of ADAMTS-5 deficient mice. We propose that in the growth plate ADAMTS-5, and not ADAMTS-4, has a physiological function in the intracellular processing of matrilins and potentially of other extracellular matrix proteins.     

A monovalent mutant of cyanovirin-N provides insight into the role of multiple interactions with gp120 for antiviral activity

Cyanovirin-N (CV-N) is a 101 amino acid cyanobacterial lectin with potent antiviral activity against HIV, mediated by high-affinity binding to branched N-linked oligomannosides on the viral surface envelope protein gp120. The protein contains two carbohydrate-binding domains, A and B, each of which binds short oligomannosides independently in vitro. The interaction to gp120 could involve either a single domain or both domains simultaneously; it is not clear which mode would elicit the antiviral activity. The model is complicated by the formation of a domain-swapped dimer form, in which part of each domain is exchanged between two monomers, which contains four functional carbohydrate-binding domains. To clarify whether multivalent interactions with gp120 are necessary for the antiviral activity, we engineered a novel mutant, P51G-m4-CVN, in which the binding site on domain A has been knocked out; in addition, a [P51G] mutation prevents the formation of domain-swapped dimers under physiological conditions. Here, we present the crystal structures at 1.8 A of the free and of the dimannose-bound forms of P51G-m4-CVN, revealing a monomeric structure in which only domain B is bound to dimannose. P51G-m4-CVN binds gp120 with an affinity almost 2 orders of magnitude lower than wt CV-N and is completely inactive against HIV. The tight binding to gp120 is recovered in the domain-swapped version of P51G-m4-CVN, prepared under extreme conditions. Our findings show that the presence of at least two oligomannoside-binding sites, either by the presence of intact domains A and B or by formation of domain-swapped dimers, is essential for activity.     

Genome-wide Association Study and Meta-Analysis Identify ISL1 as Genome-wide Significant Susceptibility Gene for Bladder Exstrophy

The bladder exstrophy-epispadias complex (BEEC) represents the severe end of the uro-rectal malformation spectrum, and is thought to result from aberrant embryonic morphogenesis of the cloacal membrane and the urorectal septum. The most common form of BEEC is isolated classic bladder exstrophy (CBE). To identify susceptibility loci for CBE, we performed a genome-wide association study (GWAS) of 110 CBE patients and 1,177 controls of European origin. Here, an association was found with a region of approximately 220kb on chromosome 5q11.1. This region harbors the ISL1 (ISL LIM homeobox 1) gene. Multiple markers in this region showed evidence for association with CBE, including 84 markers with genome-wide significance. We then performed a meta-analysis using data from a previous GWAS by our group of 98 CBE patients and 526 controls of European origin. This meta-analysis also implicated the 5q11.1 locus in CBE risk. A total of 138 markers at this locus reached genome-wide significance in the meta-analysis, and the most significant marker (rs9291768) achieved a P value of 2.13 × 10⁻¹². No other locus in the meta-analysis achieved genome-wide significance. We then performed murine expression analyses to follow up this finding. Here, Isl1 expression was detected in the genital region within the critical time frame for human CBE development. Genital regions with Isl1 expression included the peri-cloacal mesenchyme and the urorectal septum. The present study identified the first genome-wide significant locus for CBE at chromosomal region 5q11.1, and provides strong evidence for the hypothesis that ISL1 is the responsible candidate gene in this region.     

Constitutive upregulation of the transforming growth factor-β pathway in rheumatoid arthritis synovial fibroblasts

Genome-wide gene expression was comparatively investigated in early-passage rheumatoid arthritis (RA) and osteoarthritis (OA) synovial fibroblasts (SFBs; n = 6 each) using oligonucleotide microarrays; mRNA/protein data were validated by quantitative PCR (qPCR) and western blotting and immunohistochemistry, respectively. Gene set enrichment analysis (GSEA) of the microarray data suggested constitutive upregulation of components of the transforming growth factor (TGF)-β pathway in RA SFBs, with 2 hits in the top 30 regulated pathways. The growth factor TGF-β1, its receptor TGFBR1, the TGF-β binding proteins LTBP1/2, the TGF-β-releasing thrombospondin 1 (THBS1), the negative effector SkiL, and the smad-associated molecule SARA were upregulated in RA SFBs compared to OA SFBs, whereas TGF-β2 was downregulated. Upregulation of TGF-β1 and THBS1 mRNA (both positively correlated with clinical markers of disease activity/severity) and downregulation of TGF-β2 mRNA in RA SFBs were confirmed by qPCR. TGFBR1 mRNA (only numerically upregulated in RA SFBs) and SkiL mRNA were not differentially expressed. At the protein level, TGF-β1 showed a slightly higher expression, and the signal-transducing TGFBR1 and the TGF-β-activating THBS1 a significantly higher expression in RA SFBs than in OA SFBs. Consistent with the upregulated TGF-β pathway in RA SFBs, stimulation with TGF-β1 resulted in a significantly enhanced expression of matrix-metalloproteinase (MMP)-11 mRNA and protein in RA SFBs, but not in OA SFBs. In conclusion, RA SFBs show broad, constitutive alterations of the TGF-β pathway. The abundance of TGF-β, in conjunction with an augmented mRNA and/or protein expression of TGF-β-releasing THBS1 and TGFBR1, suggests a pathogenetic role of TGF-β-induced effects on SFBs in RA, for example, the augmentation of MMP-mediated matrix degradation/remodeling.     

Relative percentage and zonal distribution of mesenchymal progenitor cells in human osteoarthritic and normal cartilage

Introduction  

Mesenchymal stem cells (MSC) are highly attractive for use in cartilage regeneration. To date, MSC are usually recruited from subchondral bone marrow using microfracture. Recent data suggest that isolated cells from adult human articular cartilage, which express the combination of the cell-surface markers CD105 and CD166, are multi-potent mesenchymal progenitor cells (MPC) with characteristics similar to MSC. MPC within the cartilage matrix, the target of tissue regeneration, may provide the basis for in situ regeneration of focal cartilage defects. However, there is only limited information concerning the presence/abundance of CD105⁺/CD166⁺ MPC in human articular cartilage. The present study therefore assessed the relative percentage and particularly the zonal distribution of cartilage MPC using the markers CD105/CD166.