The blood-tendon barrier: identification and characterisation of a novel tissue barrier in tendon blood vessels.

Tissue barriers function as "gate keepers" between different compartments (usually blood and tissue) and are formed by specialised membrane-associated proteins, localising to the apicolateral plasma membrane domain of epithelial and endothelial cells. By sealing the paracellular space, the free diffusion of solutes and molecules across epithelia and endothelia is impeded. Thereby, tissue barriers contribute to the establishment and maintenance of a distinct internal and external environment, which is crucial during organ development and allows maintenance of an organ-specific homeostatic milieu. So far, various epithelial and endothelial tissue barriers have been described, including the blood-brain barrier, the blood-retina barrier, the blood-testis barrier, the blood-placenta barrier, and the cerebrospinal fluid (CSF)-brain barrier, which are vital for physiological function and any disturbance of these barriers can result in severe organ damage or even death. Here, we describe the identification of a novel barrier, located in the vascular bed of tendons, which we term the blood-tendon barrier (BTB). By using immunohistochemistry, transmission electron microscopy, and tracer studies we demonstrate the presence of a functional endothelial barrier within tendons restricting the passage of large blood-borne molecules into the surrounding tendon tissue. We further provide in vitro evidence that the BTB potentially contributes to the creation of a distinct internal tissue environment impacting upon the proliferation and differentiation of tendon-resident cells, effects which might be fundamental for the onset of tendon pathologies.

Tendons from non-diabetic humans and rats harbor a population of insulin-producing, pancreatic beta cell-like cells.

Diabetes mellitus is a risk factor for various types of tendon disorders. The mechanisms underlying diabetes associated tendinopathies remain unclear, but typically, systemic factors related to high blood glucose levels are thought to be causally involved. We hypothesize that tendon immanent cells might be directly involved in diabetic tendinopathy. We therefore analyzed human and rat tendons by immunohistochemistry, laser capture microdissection, and single cell PCR for pancreatic ß-cell associated markers. Moreover, we examined the short term effects of a single injection of streptozotocin, a toxin for GLUT2 expressing cells, in rats on insulin expression of tendon cells, and on the biomechanical properties of Achilles tendons. Tendon cells, both in the perivascular area and in the dense collagenous tissue express insulin and Glut2 on both protein and mRNA levels. In addition, glucagon and PDX-1 are present in tendon cells. Intraperitoneal injection of streptozotocin caused a loss of insulin and insulin mRNA in rat Achilles tendons after only 5 days, accompanied by a 40% reduction of mechanical strength. In summary, a so far unrecognized, extrapancreatic, insulin-producing cell type, possibly playing a major role in the pathophysiology of diabetic tendinopathy is described. In view of these data, novel strategies in tendon repair may be considered. The potential of the described cells as a tool for treating diabetes needs to be addressed by further studies.

New aspects of the molecular constituents of tissue barriers.

Epithelial and endothelial tissue barriers are based on tight intercellular contacts (Tight Junctions, TJs) between neighbouring cells. TJs are multimeric complexes, located at the most apical border of the lateral membrane. So far, a plethora of proteins locating at tight intercellular contacts have been discovered, the role of which has just partly been unraveled. Yet, there is convincing evidence that many TJ proteins exert a dual role: They act as structural components at the junctional site and they are involved in signalling pathways leading to alterations of gene expression and cell behaviour (migration, proliferation). This review will shortly summarize the classical functions of TJs and TJ-related proteins and will introduce a new category, termed the "non-classical" functions of junctional proteins. A particular focus will be directed towards the nuclear targeting of junctional proteins and the downstream effects elicited by their intranuclear activities.

The dual role of zonula occludens (ZO) proteins.

ZO (zonula occludens) proteins are scaffolding proteins providing the structural basis for the assembly of multiprotein complexes at the cytoplasmic surface of intercellular junctions. In addition, they provide a link between the integral membrane proteins and the filamentous cytoskeleton. ZO proteins belong to the large family of membrane-associated guanylate kinase (MAGUK)-like proteins comprising a number of subfamilies based on domain content and sequence similarity. Besides their structural function at cell-cell contacts, ZO proteins appear to participate in the regulation of cell growth and proliferation. Detailed molecular studies have shown that ZO proteins exhibit conserved functional nuclear localization and nuclear export motifs within their amino acid sequence. Further, ZO proteins interact with dual residency proteins localizing to the plasma membrane and the nucleus. Although the nuclear targeting of ZO proteins has well been described, many questions concerning the biological significance of this process have remained open. This review focuses on the dual role of ZO proteins, being indispensable structural components at the junctional site and functioning in signal transduction pathways related to gene expression and cell behavior.

Health status at long-term follow-up in patients treated for extremity localized Ewing Sarcoma or osteosarcoma: a Scandinavian sarcoma group study.

BACKGROUND: The purpose of this study was to evaluate late effects and symptom complaints in long-term survivors (>5 years) of Extremity Bone Sarcoma (EBS survivors). The results were compared with findings in age- and gender-matched individuals from the general population (NORMs). PATIENTS AND METHODS: Among 155 EBS survivors approached, 133 (86%) were included, and 110 of them (83%) attended an outpatient examination. Health status was evaluated by a mailed questionnaire concerning demographic and current health issues, and physical examinations at the outpatient clinic. Age- and gender-adjusted normative controls were drawn from participants of the Health Study of Nord-Trøndelag County (HUNT 2). RESULTS: Median age at follow-up was 29 (15-57) years. Median follow-up was 12 (6-22) years. Of EBS survivors 42% had > or =1 somatic disease, 33% had ototoxicity and 13% had reduced renal function. EBS survivors were more likely to have heart disease (odds ratio [OR], 7.9; 95% confidence interval [95% CI], 2.5-25.3; P = 0.001), hypertension (OR, 3.4; 95% CI, 1.1-10.1; P = 0.03) and thyroid disease (OR, 3.0; 95% CI, 1.1-8.3; P = 0.04) compared to NORMs. EBS survivors reported more diarrhoea (29% vs. 19%, P = 0.02), palpitations (23% vs. 13%, P = 0.01) and shortness of breath (11% vs. 5%, P = 0.01) than NORMs. CONCLUSIONS: EBS survivors have poorer health status compared to age- and gender-matched controls. Long-term follow-up of these patients is therefore mandatory.

Perivascular cells of the supraspinatus tendon express both tendon- and stem cell-related markers.

Tendons and ligaments are often affected by mechanical injuries or chronic impairment but other than muscle or bone they possess a low healing capacity. So far, little is known about regeneration of tendons and the role of tendon precursor cells in that process. We hypothesize that perivascular cells of tendon capillaries are progenitors for functional tendon cells and are characterized by expression of marker genes and proteins typical for mesenchymal stem cells and functional tendon cells. Immunohistochemical characterization of biopsies derived from intact human supraspinatus tendons was performed. From these biopsies perivascular cells were isolated, cultured, and characterized using RT-PCR and Western blotting. We have shown for the first time that perivascular cells within tendon tissue express both tendon- and stem/precursor cell-like characteristics. These findings were confirmed by results from in vitro studies focusing on cultured perivascular cells isolated from human supraspinatus tendon biopsies. The results suggest that the perivascular niche may be considered a source for tendon precursor cells. This study provides further information about the molecular nature and localization of tendon precursor cells, which is the basis for developing novel strategies towards tendon healing and facilitated regeneration.

Limb-sparing surgery preserves more function than amputation: a Scandinavian sarcoma group study of 118 patients.

We evaluated the long-term functional outcome in 118 patients treated for osteosarcoma or Ewing's sarcoma in the extremities a minimum of five years after treatment. We also examined if impaired function influenced their quality of life and ability to work. The function was evaluated according to the Musculoskeletal Tumor Society (MSTS) score and the Toronto Extremity Salvage Score (TESS). Quality of life was assessed by using the Short Form-36 (SF-36). The mean age at follow-up was 31 years (15 to 57) and the mean follow-up was for 13 years (6 to 22). A total of 67 patients (57%) initially had limb-sparing surgery, but four had a secondary amputation. The median MSTS score was 70% (17% to 100%) and the median TESS was 89% (43% to 100%). The amputees had a significantly lower MSTS score than those with limb-sparing surgery (p < 0.001), but there was no difference for the TESS. Tumour localisation above knee level resulted in significantly lower MSTS scores and TESS (p = 0.003 and p = 0.02, respectively). There were no significant differences in quality of life between amputees and those with limb-sparing surgery except in physical functioning. Of the patients 11% (13) did not work or study. In multivariate analysis, amputation, tumour location above the knee and having muscular pain were associated with low physical function. We conclude that most of the bone tumour survivors managed well after adjustment to their physical limitations. A total of 105 are able to work and have an overall good quality of life.

Frequent deletion of the CDKN2A locus in chordoma: analysis of chromosomal imbalances using array comparative genomic hybridisation.

The initiating somatic genetic events in chordoma development have not yet been identified. Most cytogenetically investigated chordomas have displayed near-diploid or moderately hypodiploid karyotypes, with several numerical and structural rearrangements. However, no consistent structural chromosome aberration has been reported. This is the first array-based study characterising DNA copy number changes in chordoma. Array comparative genomic hybridisation (aCGH) identified copy number alterations in all samples and imbalances affecting 5 or more out of the 21 investigated tumours were seen on all chromosomes. In general, deletions were more common than gains and no high-level amplification was found, supporting previous findings of primarily losses of large chromosomal regions as an important mechanism in chordoma development. Although small imbalances were commonly found, the vast majority of these were detected in single cases; no small deletion affecting all tumours could be discerned. However, the CDKN2A and CDKN2B loci in 9p21 were homo- or heterozygously lost in 70% of the tumours, a finding corroborated by fluorescence in situ hybridisation, suggesting that inactivation of these genes constitute an important step in chordoma development.

MyoD and Myogenin expression during myogenic phases in brown trout: a precocious onset of mosaic hyperplasia is a prerequisite for fast somatic growth.

Muscle cell recruitment (hyperplasia) during myogenesis in the vertebrate embryo is known to occur in three consecutive phases. In teleost fish (including zebrafish), however, information on myogenic precursor cell activation is largely fragmentary, and comprehensive characterization of the myogenic phases has only been fully undertaken in a single slow-growing cyprinid species by examination of MEF2D expression. Here, we use molecular techniques to provide a comprehensive characterization of MyoD and Myogenin expression during myogenic cell activation in embryos and larvae of brown trout, a fast-growing salmonid with exceptionally large embryos. Results confirm the three-phase pattern, but also demonstrate that the second and third phases begin simultaneously and progress vigorously, which is different from the previously described consecutive activation of these phases. Furthermore, we suggest that Pax7 is expressed in myogenic progenitor cells that account for second- and third-phase myogenesis. These findings are discussed in relation to teleost myotome development and to teleost growth strategies.

Treatment of local recurrences of giant cell tumour in long bones after curettage and cementing. A Scandinavian Sarcoma Group study.

We retrospectively studied local recurrence of giant cell tumour in long bones following treatment with curettage and cementing in 137 patients. The median follow-up time was 60 months (3 to 166). A total of 19 patients (14%) had at least one local recurrence, the first was diagnosed at a median of 17 months (3 to 29) after treatment of the primary tumour. There were 13 patients with a total of 15 local recurrences who were successfully treated by further curettage and cementing. Two patients with a second local recurrence were consequently treated twice. At the last follow-up, at a median of 53 months (3 to 128) after the most recent operation, all patients were free from disease and had good function. We concluded that local recurrence of giant cell tumour after curettage and cementing in long bones can generally be successfully treated with further curettage and cementing, with only a minor risk of increased morbidity. This suggests that more extensive surgery for the primary tumour in an attempt to obtain wide margins is not the method of choice, since it leaves the patient with higher morbidity with no significant gain with respect to cure of the disease.

Surgical treatment of skeletal metastatic lesions of the proximal femur: endoprosthesis or reconstruction nail?

We report positive and negative factors associated with the most commonly-used methods of reconstruction after pathological fracture of the proximal femur. The study was based on 142 patients treated surgically for 146 metastatic lesions between 1996 and 2003. The local rate of failure was 10.3% (15 of 146). Of 37 operations involving osteosynthetic devices, six failed (16.2%) compared with nine (8.3%) in 109 operations involving endoprostheses. Of nine cases of prosthetic failure, four were due to periprosthetic fractures and three to recurrent dislocation. In the osteosynthesis group, three (13.6%) of 22 reconstruction nails failed. The two-year risk of re-operation after any type of osteosynthesis was 0.35 compared with 0.18 after any type of endoprosthetic reconstruction (p = 0.07). Endoprosthetic reconstructions are preferable to the use of reconstruction nails and other osteosynthetic devices for the treatment of metastatic lesions in the proximal third of the femur.

In search of the astrocytic factor(s) modulating blood-brain barrier functions in brain capillary endothelial cells in vitro.

(1) The blood-brain barrier (BBB) is formed by brain capillary endothelial cells (ECs). There are various cell types, in particular astrocytes, but also pericytes and neurons, located in close vicinity to the capillary ECs which may influence formation and function of the BBB. Based on this consideration, this paper discusses various aspects of the influence of the surrounding cells on brain capillary ECs with special focus on the role of astrocytes. (2) Based on the morphology of the BBB, important aspects of brain EC functions are summarized, such as transport functions and maintenance of low paracellular permeability. Moreover, various facets are discussed with respect to the influence of astrocytes, pericytes, microglia, and neurons on the BBB. Data on the role of glial cells in the ontogenesis of the BBB are presented subsequently. The knowledge on this subject is far from being complete, however, these data imply that the neural/neuronal environment rather than glial cells may be of importance in the maturation of the barrier. (3) The role of glial cells in the induction and maintenance of the BBB is discussed under physiological as well as pathological conditions. Although the literature presents manifold evidence for a great variety of effects induced by astroglia, there are also many controversies, which may result from different cellular models and experimental conditions used in the respective studies. Numerous factors secreted by astrocytes have been shown to induce a BBB phenotype. On the molecular level, increased expression of barrier-relevant proteins (e.g., tight junction proteins) is documented in the presence of astrocyte-derived factors, and many studies demonstrate the improvement of physiological parameters, such as increased transendothelial resistance and decreased paracellular permeability, in different in vitro models of the BBB. Moreover, one has to take into account that the interaction of brain ECs and astrocytes is bi-directional, and that the other cell types surrounding the brain microvasculature also contribute to BBB function or dysfunction, respectively. (4) In conclusion, it is expected that the present and future research focused on molecular mechanisms and signaling pathways will produce new and exciting insights into the complex network of BBB regulation: the cornerstone is laid.

Accuracy of cytology for diagnosis of lipomatous tumors: comparison with magnetic resonance and computed tomography findings in 175 cases.

PURPOSE: To assess the value of fine-needle aspiration cytology in the diagnostic work-up of lipomatous tumors of the extremities and trunk, and to identify specific radiological features that could aid in the preoperative evaluation. MATERIAL AND METHODS: 175 patients with subfascial lipomatous tumors who had undergone preoperative magnetic resonance imaging or computed tomography and fine-needle aspiration cytology were studied. The percentage of fat within the lesion was visually graded from the images as: none, 1-75%, 75-95%, or 95-100%. The histological and cytological diagnoses were compared and in discordant cases the radiological images were re-reviewed. RESULTS: There was cytological and histological concordance in 96% of lipomas and in 85% of atypical lipomatous tumors (ALT) and liposarcomas. Most discordant cases exhibited 1-75% fat. Radiological review suggested that cytological sampling problems due to tumor heterogeneity were the main cause of diagnostic difficulties. The majority of tumors with less than 75% fat were liposarcomas, and in no liposarcoma was the fat content higher than 75%. Both ALT and lipoma were found in the 95-100% group. CONCLUSION: Cytology can be highly accurate in the diagnosis of lipomatous tumors, including ALT; however, critical comparison with the radiological findings increases diagnostic security. In tumors with fat content visually assessed as less than 75% of the tumor volume, liposarcoma is the most likely diagnosis and a cytological diagnosis of ALT or lipoma should be questioned. In lesions with 75-95% fat, liposarcoma is unlikely, but FNAC is still indicated for safety. In lesions with 95-100% fat, FNAC is only indicated if the differentiation between lipoma and ALT influences the treatment strategy.

Oxidative stress in cultured cerebral endothelial cells induces chromosomal aberrations, micronuclei, and apoptosis.

There is evidence accumulating that brain microvasculature is involved critically in oxidative stress-mediated brain damage. Cultured cerebral microvascular endothelial cells were used to demonstrate the cytotoxic and genotoxic effects elicited by hypoxia/reoxygenation and DMNQ treatment in vitro. In addition, the effect of glucose deprivation during oxidative insult was assessed. The parameters determined were: 1) chromosomal aberrations; 2) induction of micronuclei; and 3) apoptosis. Our results indicate that both the exposure of the cerebral endothelial cells to 24 hr of hypoxia followed by 4 hr of reoxygenation, and treatment with the redox cycling quinone DMNQ, increased markedly the occurrence of chromosomal aberrations and micronuclei. It was found that expression of p53 was induced by oxidative stress, particularly when glucose had been omitted from the culture medium. Aglycemic culture conditions in general exacerbated the cytotoxic effects of oxidative insults, as evidenced by the increase in apoptotic cells and the decrease in the mitotic index. Interestingly, neither an elevation of cell lysis nor an increase in necrosis has been observed during our experiments. In summary, our data indicate that oxidative stress exerts considerable genotoxic and cytotoxic effects on cerebral endothelial cells, which might contribute to the progression of tissue damage in the central nervous system.

Identification and localization of M-CoREST (1A13), a mouse homologue of the human transcriptional co-repressor CoREST, in the developing mouse CNS.

By means of subtractive and differential hybridization techniques we have identified a novel murine gene (1A13) the expression of which is developmentally regulated in the mouse brain. Comparison of the nucleotide and predicted protein sequence revealed closest relationship of 1A13 to human CoREST, a transcriptional co-repressor required for regulation of neural-specific gene expression. Thus, we will refer to 1A13 as M-CoREST. As shown by in situ hybridization and Northern blotting, expression of M-CoREST mRNA is abundant in neural tissue at early embryonic stages but declines significantly towards birth, coincident with the progression of CNS maturation.