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.

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.

The distribution of membrane-bound 14-3-3 proteins in organelle-enriched fractions of germinating lily pollen.

Proteins of the 14-3-3 family show a broad range of activities in plants, depending on their localisation in different cellular compartments. Different organelle membranes of pollen grains and pollen tubes of Lilium longiflorum Thunb. were separated simultaneously using optimised discontinuous sucrose density centrifugation. The obtained organelle-enriched fractions were identified as vacuolar, Golgi, endoplasmic reticulum and plasma membranes, according to their marker enzyme activities, and were assayed for membrane-bound 14-3-3 proteins by immunodetection. 14-3-3 proteins were detected in the cytoplasm as well as in all obtained organelle fractions but were also released into the extracellular medium. In pollen grains, much more plasma membrane-bound 14-3-3 proteins were detected than in the PM-enriched fraction of pollen tubes, whereas the level of Golgi- and ER-associated 14-3-3 proteins was similar in pollen grains and tubes. This shift in the localisation of membrane-associated 14-3-3 proteins is probably correlated with a change in the major function of 14-3-3 proteins, e.g., perhaps changing from initiating pollen grain germination by activation of the PM H +-ATPase to recruitment of membrane proteins via the secretory pathway during tube elongation.

Heterogeneity of the vacuolar pyrophosphatase protein from Chenopodium rubrum.

Activities of the tonoplast ATPase (V-ATPase EC 3.6.1.3) and PPase (V-PPase EC 3.6.1.1) provide the proton gradient driving the accumulation of various metabolites, organic and inorganic ions in the plant vacuole. We used anion exchange chromatography, liquid-phase isoelectric focusing (IEF), and continuous-elution native polyacrylamide gel electrophoresis (preparative PAGE) to enrich the V-PPase from solubilized tonoplast proteins from suspension cultured cells of Chenopodium rubrum L.The fractions were identified by their enzymatic activity, sensitivity towards the specific PPase inhibitor aminomethylenediphosphonate, apparent molecular weight, and immunological reactivity with an antibody raised against mung bean V-PPase. All these different methods used for the separation of solubilized tonoplast proteins revealed the existence of two physically separable V-PPase proteins exhibiting substrate specific enzymatic activity and 66 kDa apparent molecular weight after sodium dodecyl sulfate(SDS)-PAGE. The isoelectric points of the active V-PPase forms were 5.05 and 5.48 (V-ATPase 6.1). On the basis of the observation of high recoveries of enzymatic activity after different preparations we suggest that the V-PPase proteins separated may represent physiologically occurring forms of the enzyme which cannot be distinguished by SDS-PAGE and Western blot.

Molecular and physiological characterisation of a 14-3-3 protein from lily pollen grains regulating the activity of the plasma membrane H+ ATPase during pollen grain germination and tube growth.

A 14-3-3 protein has been cloned and sequenced from a cDNA library constructed from mRNAs of mature pollen grains of Lilium longiflorum Thunb. Monoclonal antibodies (MUP 5 or MUP 15) highly specific against 14-3-3 proteins recognised a 30-kDa protein in the cytoplasmic fraction of many various lily tissues (leaves, bulbs, stems, anther filaments, pollen grains, stigmas) and in other plants (Arabidopsis seedlings, barley recombinant 14-3-3). In addition, 14-3-3 proteins were detected in a microsomal fraction isolated from pollen grains and tubes, and the amount of membrane-bound 14-3-3 proteins as well as the amount of the plasma membrane (PM) H+ ATPase increased during germination of pollen grains and tube growth. No change was observed in the cytoplasmic fraction. A further increase in the amount of 14-3-3 proteins in the microsomal fraction was observed when pollen grains were incubated in germination medium containing 1 microM fusicoccin (FC) whereas the number of 14-3-3s in the cytoplasmic fraction decreased. Fusicoccin also protected membrane-bound 14-3-3 proteins from dissociation after washing with the chaotropic salt KI. Furthermore, FC stimulated the PM H+ ATPase activity, the germination frequency and the growth rate of pollen tubes, thus indicating that a modulation of the PM H+ ATPase activity by interaction with 14-3-3 proteins may regulate germination and tube growth of lily pollen.