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Vascular endothelial growth factor (VEGF) signaling is crucial for a large variety of cellular processes, not only related to angiogenesis but also in nonvascular cell types. We have previously shown that controlling angiogenesis by reducing VEGF-A signaling positively affects tendon healing. We now hypothesize that VEGF signaling in non-endothelial cells may contribute to tendon pathologies. By immunohistochemistry we show that VEGFR1, VEGFR2, and VEGFR3 are expressed in murine and human tendon cells in vivo. In a rat Achilles tendon defect model we show that VEGFR1, VEGFR3, and VEGF-D expression are increased after injury. On cultured rat tendon cells we show that VEGF-D stimulates cell proliferation in a dose-dependent manner; the specific VEGFR3 inhibitor SAR131675 reduces cell proliferation and cell migration. Furthermore, activation of VEGFR2 and -3 in tendon-derived cells affects the expression of mRNAs encoding extracellular matrix and matrix remodeling proteins. Using explant model systems, we provide evidence, that VEGFR3 inhibition prevents biomechanical deterioration in rat tail tendon fascicles cultured without load and attenuates matrix damage if exposed to dynamic overload in a bioreactor system. Together, these results suggest a strong role of tendon cell VEGF signaling in mediation of degenerative processes. These findings give novel insight into tendon cell biology and may pave the way for novel treatment options for degenerative tendon diseases.

Summary: Background: Treatment of degenerating tendons still presents a major challenge, since the aetiology of tendinopathies remains poorly understood. Besides mechanical overuse, further known predisposing factors include rheumatoid arthritis, diabetes, obesity or smoking all of which combine with a systemic inflammation. Methods: To determine whether the systemic inflammation accompanying these conditions contributes to the onset of tendinopathy, we studied the effect of a systemic inflammation induced by an allergic episode on tendon properties. To this end, we induced an allergic response in mice by exposing them to a timothy grass pollen allergen and subsequently analysed both their flexor and Achilles tendons. Additionally, we analysed data from a health survey comprising data from more than 10.000 persons for an association between the occurrence of an allergy and tendinopathy. Findings: Biomechanical testing and histological analysis revealed that tendons from allergic mice not only showed a significant reduction of both elastic modulus and tensile stress, but also alterations of the tendon matrix. Moreover, treatment of 3D tendon-like constructs with sera from allergic mice resulted in a matrix-remodelling expression profile and the expression of macrophage-associated markers and matrix metalloproteinase 2 (MMP2) was increased in allergic Achilles tendons. Data from the human health study revealed that persons suffering from an allergy have an increased propensity to develop a tendinopathy. Interpretation: Our study demonstrates that the presence of a systemic inflammation accompanying an allergic condition negatively impacts on tendon structure and function. Funding: This study was financially supported by the Fund for the Advancement of Scientific Research at Paracelsus Medical University (PMU-FFF E-15/22/115-LEK), by the Land Salzburg, the Salzburger Landeskliniken (SALK, the Health Care Provider of the University Hospitals Landeskrankenhaus and Christian Doppler Klinik), the Paracelsus Medical University, Salzburg and by unrestricted grants from Bayer, AstraZeneca, Sanofi-Aventis, Boehringer-Ingelheim.

MicroRNAs (miRNAs) have emerged as key regulators orchestrating a wide range of inflammatory and fibrotic diseases. However, the role of miRNAs in degenerative shoulder joint disorders is poorly understood. The aim of this explorative case‐control study was to identify pathology‐related, circulating miRNAs in patients with chronic rotator cuff tendinopathy and degenerative rotator cuff tears (RCT). In 2017, 15 patients were prospectively enrolled and assigned to three groups based on the diagnosed pathology: (i) no shoulder pathology, (ii) chronic rotator cuff tendinopathy, and (iii) degenerative RCTs. In total, 14 patients were included. Venous blood samples (“liquid biopsies”) were collected from each patient and serum levels of 187 miRNAs were determined. Subsequently, the change in expression of nine candidate miRNAs was verified in tendon biopsy samples, collected from patients who underwent arthroscopic shoulder surgery between 2015 and 2018. Overall, we identified several miRNAs to be progressively deregulated in sera from patients with either chronic rotator cuff tendinopathy or degenerative RCTs. Importantly, for the several of these miRNAs candidates repression was also evident in tendon biopsies harvested from patients who were treated for a supraspinatus tendon tear. As similar expression profiles were determined for tendon samples, the newly identified systemic miRNA signature has potential as novel diagnostic or prognostic biomarkers for degenerative rotator cuff pathologies. © 2019 The Authors. Journal of Orthopaedic Research ® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. Inc. J Orthop Res 38:202–211, 2020

Angiogenesis, the process of new blood vessel formation from existing blood vessels, is a key aspect of virtually every repair process. During wound healing an extensive, but immature and leaky vascular plexus forms which is subsequently reduced by regression of non-functional vessels. More recent studies indicate that uncontrolled vessel growth or impaired vessel regression as a consequence of an excessive inflammatory response can impair wound healing, resulting in scarring and dysfunction. However, in order to elucidate targetable factors to promote functional tissue regeneration we need to understand the molecular and cellular underpinnings of physiological angiogenesis, ranging from induction to resolution of blood vessels. Especially for avascular tissues (e.g. cornea, tendon, ligament, cartilage, etc.), limiting rather than boosting vessel growth during wound repair potentially is beneficial to restore full tissue function and may result in favourable long-term healing outcomes.

Tendons and tendon interfaces have a very limited regenerative capacity, rendering their injuries clinically challenging to resolve. Tendons sense muscle-mediated load; however, our knowledge on how loading affects tendon structure and functional adaption remains fragmentary. Here, we provide evidence that the matricellular protein secreted protein acidic and rich in cysteine (SPARC) is critically involved in the mechanobiology of tendons and is required for tissue maturation, homeostasis, and enthesis development. We show that tendon loading at the early postnatal stage leads to tissue hypotrophy and impaired maturation of Achilles tendon enthesis in Sparc -/- mice. Treadmill training revealed a higher prevalence of spontaneous tendon ruptures and a net catabolic adaptation in Sparc -/- mice. Tendon hypoplasia was attenuated in Sparc -/- mice in response to muscle unloading with botulinum toxin A. In vitro culture of Sparc -/- three-dimensional tendon constructs showed load-dependent impairment of ribosomal S6 kinase activation, resulting in reduced type I collagen synthesis. Further, functional calcium imaging revealed that lower stresses were required to trigger mechanically induced responses in Sparc -/- tendon fascicles. To underscore the clinical relevance of the findings, we further demonstrate that a missense mutation (p.Cys130Gln) in the follistatin-like domain of SPARC, which causes impaired protein secretion and type I collagen fibrillogenesis, is associated with tendon and ligament injuries in patients. Together, our results demonstrate that SPARC is a key extracellular matrix protein essential for load-induced tendon tissue maturation and homeostasis.

Tendon disorders frequently occur and recent evidence has clearly implicated the presence of immune cells and inflammatory events during early tendinopathy. However, the origin and properties of these cells remain poorly defined. Therefore, the aim of this study was to determine the presence of cells in healthy rodent and human tendon tissue fulfilling macrophage-like functions. Using various transgenic reporter mouse models, we demonstrate the presence of tendon-resident cells in the dense matrix of the tendon core expressing the fractalkine (Fkn) receptor CX3CR1 and its cognate ligand CX3CL1/Fkn. Pro-inflammatory stimulation of 3D tendon-like constructs in vitro resulted in a significant increase in the expression of IL-1β, IL-6, Mmp3, Mmp9, CX3CL1 and epiregulin, which has been reported to contribute to inflammation, wound healing and tissue repair. Furthermore, we demonstrate that inhibition of the Fkn receptor blocked tendon cell migration in vitro, and show the presence of CX3CL1/CX3CR1/EREG-expressing cells in healthy human tendons. Taken together, we demonstrate the presence of CX3CL1+/CX3CR1+ ‘tenophages’ within the healthy tendon proper, which potentially fulfill surveillance functions in tendons. This article has an associated First Person interview with the first author of the paper., Summary: Here, we demonstrate the presence of a macrophage-like, CX3CL1/CX3CR1-expressing tendon cell population within the healthy tendon proper, which potentially fulfills a surveillance function.

Background/Aims: Effective wound-healing generally requires efficient re-vascularization after injury, ensuring sufficient supply with oxygen, nutrients, and various cell populations. While this applies to most tissues, tendons are mostly avascular in nature and harbor relatively few cells, probably contributing to their poor regenerative capacity. Considering the minimal vascularization of healthy tendons, we hypothesize that controlling angiogenesis in early tendon healing is beneficial for repair tissue quality and function. Methods: To address this hypothesis, Bevacizumab, a monoclonal antibody blocking VEGF-A signaling, was locally injected into the defect area of a complete tenotomy in rat Achilles tendon. At 28 days post-surgery, the defect region was investigated using immunohistochemistry against vascular and lymphatic epitopes. Polarization microscopy and biomechanical testing was used to determine tendon integrity and gait analysis for functional testing in treated vs non-treated animals. Results: Angiogenesis was found to be significantly reduced in the Bevacizumab treated repair tissue, accompanied by significantly reduced cross sectional area, improved matrix organization, increased stiffness and Young’s modulus, maximum load and stress. Further, we observed an improved gait pattern when compared to the vehicle injected control group. Conclusion: Based on the results of this study we propose that reducing angiogenesis after tendon injury can improve tendon repair, potentially representing a novel treatment-option.

Tendon disorders frequently occur and recent evidence has clearly implicated the presence of immune cells and inflammatory events during early tendinopathy. However, the origin and properties of these cells remain poorly defined. Therefore, the aim of this study was to determine the presence of myleoid cells in healthy rodent and human tendon tissue and to characterize them. Using various transgenic reporter mouse models, we demonstrate the presence of tendon cells in the dense matrix of the tendon core expressing the fractalkine (Fkn) receptor CX3CR1 and its cognate ligand CX3CL1/Fkn. Pro-inflammatory stimulation of 3D tendon-like constructsin vitroresulted in a significant increase in the expression of IL-1β, IL-6, Mmp3, Mmp9, Cx3cl1, and epiregulin which has been reported to contribute to inflammation, wound healing, and tissue repair. Furthermore, we demonstrate that inhibition of the fractalkine receptor blocked tendon cell migrationin vitroand show the presence of CX3CR1/CX3CL1/EREG expressing cells in healthy human tendons. Taken together, we demonstrate the presence of CX3CL1+/CX3CR1+ “tenophages” within the healthy tendon proper potentially fulfilling surveillance functions in tendons.Summary StatementHere, we demonstrate the presence of a macrophage-like, CX3CL1/CX3CR1-expressing cell population within the healthy tendon proper potentially fulfilling a surveillance function.

Tendinopathy is accompanied by a cascade of inflammatory events promoting tendon degeneration. Among various cytokines, interleukin-1&beta, plays a central role in driving catabolic processes, ultimately resulting in the activation of matrix metalloproteinases and a diminished collagen synthesis, both of which promote tendon extracellular matrix degradation. Pulsed electromagnetic field (PEMF) therapy is often used for pain management, osteoarthritis, and delayed wound healing. In vitro PEMF treatment of tendon-derived cells was shown to modulate pro-inflammatory cytokines, potentially limiting their catabolic effects. However, our understanding of the underlying cellular and molecular mechanisms remains limited. We therefore investigated the transcriptome-wide responses of Il-1&beta, primed rat Achilles tendon cell-derived 3D tendon-like constructs to high-energy PEMF treatment. RNASeq analysis and gene ontology assignment revealed various biological processes to be affected by PEMF, including extracellular matrix remodeling and negative regulation of apoptosis. Further, we show that members of the cytoprotective Il-6/gp130 family and the Il-1&beta, decoy receptor Il1r2 are positively regulated upon PEMF exposure. In conclusion, our results provide fundamental mechanistic insight into the cellular and molecular mode of action of PEMF on tendon cells and can help to optimize treatment protocols for the non-invasive therapy of tendinopathies.

Although several studies revealed a multifactorial pathogenesis of degenerative rotator cuff disorders, the impact and interaction of extrinsic variables is still poorly understood. Thus, this study aimed at uncovering the effect of patient‐ and pathology‐specific risk factors that may contribute to degeneration of the rotator cuff tendons. Between 2015 and 2018, 54 patients who underwent arthroscopic shoulder surgery at three specialized shoulder clinics were prospectively included. Using tendon samples harvested from the macroscopically intact subscapularis (SSC) tendon, targeted messenger RNA expression profile analysis was performed in the first cohort (n = 38). Furthermore, histological analyses were conducted on tendon tissue samples obtained from a second cohort (n = 16). Overall, both study cohorts were comparable concerning patient demographics. Results were then analyzed with respect to specific extrinsic factors, such as patient age, body mass index, current as well as previous professions and sport activities, smoking habit, and systemic metabolic diseases. While patient age, sports‐activity level, and preexisting rotator cuff lesions were considered to contribute most strongly to tendinopathogenesis, no further coherences were found. With regards to gene expression analysis, change in expression correlated most strongly with patient age and severity of the rotator cuff pathology. Further, chronic disorders increased overall gene expression variation. Taken together, our study provides further evidence that tendon degeneration is the consequence of a multifactorial process and pathological changes of the supraspinatus tendon affect the quality of SSC tendon and most likely vice versa. Therefore, the rotator cuff tendons need to be considered as a unit when managing rotator cuff pathologies. © 2019 The Authors. Journal of Orthopaedic Research ® published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society J Orthop Res 38:182–191, 2020

Tendons harbor various cell populations, including cells displaying classical adult mesenchymal stromal cell criteria. Previous studies have shown that a tenogenic phenotype is more effectively maintained in a 3D cell culture model under mechanical load. This chapter describes a method to isolate tendon-derived cells from rat Achilles tendons and the subsequent formation of 3D-embedded cell cultures. These tendon-like constructs can then be analyzed by various means, including histology, immunohistochemistry, qPCR, or standard protein analysis techniques.

Tendons harbor various cell populations, including cells displaying classical adult mesenchymal stromal cell criteria. Previous studies have shown that a tenogenic phenotype is more effectively maintained in a 3D cell culture model under mechanical load. This chapter describes a method to isolate tendon-derived cells from rat Achilles tendons and the subsequent formation of 3D-embedded cell cultures. These tendon-like constructs can then be analyzed by various means, including histology, immunohistochemistry, qPCR, or standard protein analysis techniques.

Highlights • Establishment of an alternative method beside routinely used ELISA to measure levels of sAxl and Gas6 in serological material of HGG patients is proposed. • Both antibodies are established with the powerful combination of protein detection and PCR amplification using the Proseek Single-Plex Assay. • This tool quantifies single proteins in solution with a maximum of sensitivity and specificity to visualize low levels of proteins in just 1 μl blood sample within one day., • The receptor tyrosine kinase (RTK) Axl and its ligand Gas6 are critically involved in the pathogenesis of high-grade glioma (HGG). Both proteins were found to be overexpressed e.g. in tumor cells, mediating cell proliferation and migration as well as tumor angiogenesis and neuroinflammation. The extracellular domain of Axl (sAxl) and Gas6 were found in the peri-tumoral edema and blood of animals as well as in human glioma tissue. Therefore, we monitored the level of sAxl and Gas6 in human blood samples. To increase the sensitivity of protein detection beyond commonly used standard methods we preliminary tested the innovative Proseek Single-Plex Protein Assay Kit System from Olink Bioscience together with new antibodies against the soluble RTK sAxl and its ligand Gas6. We conclude that the Proseek method is a highly sensitive and fast procedure that can be used as a possible powerful tool compared to routinely used ELISA-methods.

Despite significant advancements in bone tissue-engineering applications, the clinical impact of bone marrow stromal cells (BMSCs) for the treatment of large osseous defects remains limited. Therefore, other cell sources are under investigation for their osteogenic potential to repair bone. In this study, tendon-derived stromal cells (TDSCs) were evaluated in comparison to BMSCs to support the functional repair of a 5 mm critical-sized, segmental defect in the rat femur. Analysis of the trilineage differentiation capacity of TDSCs and BMSCs cultured on collagen sponges revealed impaired osteogenic differentiation and mineral deposition of TDSCs in vitro, whereas chondrogenic and adipogenic differentiation was evident for both cell types. Radiographic assessment demonstrated that neither cell type significantly improved the healing rate of a challenging 5 mm segmental femoral defect. Transplanted TDSCs and BMSCs both led to the formation of only small amounts of bone in the defect area, and histological evaluation revealed non-mineralized, collagen-rich scar tissue to be present within the defect area. Newly formed lamellar bone was restricted to the defect margins, resulting in closure of the medullary cavity. Interestingly, in comparison to BMSCs, significantly more TDSC-derived cells were present at the osteotomy gap up to 8 weeks after transplantation and were also found to be located within newly formed lamellar bone, suggesting their capacity to directly contribute to de novo bone formation. To our knowledge, this is the first study investigating the in vivo capacity of TDSCs to regenerate a critical-sized defect in the rat femur. Copyright © 2015 John Wiley & Sons, Ltd.

Chronic and acute tendinopathies are difficult to treat and tendon healing is generally a very slow and incomplete process and our general understanding of tendon biology and regeneration lags behind that of muscle or bone. Although still largely unexplored, several studies suggest a positive effect of nutritional interventions on tendon health and repair. With this study, we aim to reveal effects of a high-glucose diet on tendon neoformation in a non-diabetic rat model of Achilles tenotomy. After surgery animals received either a high-glucose diet or a control diet for 2 and 4 weeks, respectively. Compared to the control group, tendon repair tissue thickness and stiffness were increased in the high-glucose group after 2 weeks and gait pattern was altered after 1 and 2 weeks. Cell proliferation was up to 3-fold higher and the expression of the chondrogenic marker genes Sox9, Col2a1, Acan and Comp was significantly increased 2 and 4 weeks post-surgery. Further, a moderate increase in cartilage-like areas within the repair tissue was evident after 4 weeks of a high-glucose diet regimen. In summary, we propose that a high-glucose diet significantly affects tendon healing after injury in non-diabetic rats, potentially driving chondrogenic degeneration.

Acute and chronic tendinopathies remain clinically challenging and tendons are predisposed to degeneration or injury with age. Despite the high prevalence of tendon disease in the elderly, our current understanding of the mechanisms underlying the age-dependent deterioration of tendon function remains very limited. Here, we show that Secreted protein acidic and rich in cysteine (Sparc) expression significantly decreases in healthy-aged mouse Achilles tendons. Loss of Sparc results in tendon collagen fibrillogenesis defects and Sparc−/− tendons are less able to withstand force in comparison with their respective wild type counterparts. On the cellular level, Sparc-null and healthy-aged tendon-derived cells exhibited a more contracted phenotype and an altered actin cytoskeleton. Additionally, an elevated expression of the adipogenic marker genes PPARγ and Cebpα with a concomitant increase in lipid deposits in aged and Sparc−/− tendons was observed. In summary, we propose that Sparc levels in tendons are critical for proper collagen fibril maturation and its age-related decrease, together with a change in ECM properties favors lipid accretion in tendons.

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 β-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.

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.

Monoclonal antibodies against the K+ channel KAT1 of Arabidopsis thaliana, a low abundance, plant plasma membrane protein, were generated by genetic immunisation to avoid the time and labour consuming purification of native or recombinant proteins and peptides usually necessary for conventional immunisation techniques. The resulting polyclonal and monoclonal antibody sera recognised a single protein band in a microsomal fraction of wild-type A. thaliana leaves and in membrane fractions of transgenic yeast cells and tobacco plants expressing the KAT1 protein. Therefore, genetic immunisation is suitable for generating monoclonal antibodies against plant proteins and particularly, against plant membrane proteins of low abundance.

Despite significant advancements in bone tissue-engineering applications, the clinical impact of bone marrow stromal cells (BMSCs) for the treatment of large osseous defects remains limited. Therefore, other cell sources are under investigation for their osteogenic potential to repair bone. In this study, tendon-derived stromal cells (TDSCs) were evaluated in comparison to BMSCs to support the functional repair of a 5 mm critical-sized, segmental defect in the rat femur. Analysis of the trilineage differentiation capacity of TDSCs and BMSCs cultured on collagen sponges revealed impaired osteogenic differentiation and mineral deposition of TDSCs in vitro, whereas chondrogenic and adipogenic differentiation was evident for both cell types. Radiographic assessment demonstrated that neither cell type significantly improved the healing rate of a challenging 5 mm segmental femoral defect. Transplanted TDSCs and BMSCs both led to the formation of only small amounts of bone in the defect area, and histological evaluation revealed non-mineralized, collagen-rich scar tissue to be present within the defect area. Newly formed lamellar bone was restricted to the defect margins, resulting in closure of the medullary cavity. Interestingly, in comparison to BMSCs, significantly more TDSC-derived cells were present at the osteotomy gap up to 8 weeks after transplantation and were also found to be located within newly formed lamellar bone, suggesting their capacity to directly contribute to de novo bone formation. To our knowledge, this is the first study investigating the in vivo capacity of TDSCs to regenerate a critical-sized defect in the rat femur. Copyright © 2015 John WileySons, Ltd.

To maintain the precise internal milieu of the mammalian central nervous system, well-controlled transfer of molecules from periphery into brain is required. Recently the soluble and cell-surface albumin-binding glycoprotein SPARC (secreted protein acidic and rich in cysteine) has been implicated in albumin transport into developing brain, however the exact mechanism remains unknown. We postulate that SPARC is a docking site for albumin, mediating its uptake and transfer by choroid plexus epithelial cells from blood into cerebrospinal fluid (CSF). We used in vivo physiological measurements of transfer of endogenous (mouse) and exogenous (human) albumins, in situ Proximity Ligation Assay (in situ PLA), and qRT-PCR experiments to examine the cellular mechanism mediating protein transfer across the bloodCSF interface. We report that at all developmental stages mouse albumin and SPARC gave positive signals with in situ PLAs in plasma, CSF and within individual plexus cells suggesting a possible molecular interaction. In contrast, in situ PLA experiments in brain sections from mice injected with human albumin showed positive signals for human albumin in the vascular compartment that were only rarely identifiable within choroid plexus cells and only at older ages. Concentrations of both endogenous mouse albumin and exogenous (intraperitoneally injected) human albumin were estimated in plasma and CSF and expressed as CSF/plasma concentration ratios. Human albumin was not transferred through the mouse bloodCSF barrier to the same extent as endogenous mouse albumin, confirming results from in situ PLA. During postnatal development Sparc gene expression was higher in early postnatal ages than in the adult and changed in response to altered levels of albumin in blood plasma in a differential and developmentally regulated manner. Here we propose a possible cellular route and mechanism by which albumin is transferred from blood into CSF across a sub-population of specialised choroid plexus epithelial cells. HEALTH-F2-2009-241778

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.

Combining the patch-clamp method with single-cell reverse transcription polymerase chain reaction (scRT-PCR) a fusicoccin-induced current reflecting the activity of the plasma membrane H a ATPase of lily pollen protoplasts was measured and subsequently, the ATPase-encoding mRNAs were collected and amplified. Southern blot signals were observed in all 'patch^ catch' experiments and could be detected even in 2560-fold dilutions of the pollen contents. H a ATPase mRNAs were detectable only in the vegetative but not in the generative cell of pollen as confirmed by immunolocalisation. In 15% of the scRT- PCR experiments, a random non-reproducibility of the PCR was observed, probably caused by varying amounts of ATPase mRNAs in the protoplasts. fl 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

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.

Background: Toxicity of the local anesthetic bupivacaine (BV) has been a matter of debate across medical fields. Numerous in vitro studies demonstrate considerable toxicity of BV on various cell types. Purpose: This study addresses the question of how tendon tissue responds to BV in vivo and in vitro. Study Design: Controlled laboratory study. Methods: In vitro studies on cultured rat Achilles tendon–derived cells were performed with cell viability assays and cleaved caspase 3 immunocytochemistry. Quantitative reverse transcription–polymerase chain reaction, Western blotting, gelatin zymography, and a biomechanical testing routine were applied on rat Achilles tendons at 1 and 4 weeks after a single unilateral peritendinous injection of 0.5% BV. The BV-mediated cell death in tendons was estimated with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and immunohistochemical detection of cleaved caspase 3. Results: Treatment of rat tendon–derived cells with 0.5% bupivacaine for 10 minutes had detrimental effects on cell viability, which can be reduced by N-acetyl-L-cysteine or reduction of extracellular calcium. In vivo, single peritendinous injections of BV caused apoptosis in endotenon cells and an increase of pro–matrix metalloproteinase-9 after 6 hours. The collagen ratio shifted toward collagen type III after 6 hours and 2 days; scleraxis messenger RNA (mRNA) expression was reduced by 87%. Maximum tensile load was reduced by 17.6% after 1 week. Conclusion: Bupivacaine exerts a severe, reactive oxygen species–mediated effect on tendon cell viability in vitro in a time- and dose-dependent manner, depending on extracellular calcium concentration. Culture conditions need to be taken into account when in vitro data are translated into the in vivo situation. In vivo, administration of BV elicits a marked but temporary functional damage. Clinical Relevance: Local anesthetics cause short-term alterations in rat tendons, which, if occurring in humans to a similar extent, may be relevant regarding decreased biomechanical properties and increased vulnerability to tendon overload or injury.

Exchange mechanisms across the blood–cerebrospinal fluid (CSF) barrier in the choroid plexuses within the cerebral ventricles control access of molecules to the central nervous system, especially in early development when the brain is poorly vascularised. However, little is known about their molecular or developmental characteristics. We examined the transcriptome of lateral ventricular choroid plexus in embryonic day 15 (E15) and adult mice. Numerous genes identified in the adult were expressed at similar levels at E15, indicating substantial plexus maturity early in development. Some genes coding for key functions (intercellular/tight junctions, influx/efflux transporters) changed expression during development and their expression patterns are discussed in the context of available physiological/permeability results in the developing brain. Three genes: Secreted protein acidic and rich in cysteine (Sparc), Glycophorin A (Gypa) and C (Gypc), were identified as those whose gene products are candidates to target plasma proteins to choroid plexus cells. These were investigated using quantitative- and single-cell-PCR on plexus epithelial cells that were albumin- or total plasma protein-immunopositive. Results showed a significant degree of concordance between plasma protein/albumin immunoreactivity and expression of the putative transporters. Immunohistochemistry identified SPARC and GYPA in choroid plexus epithelial cells in the embryo with a subcellular distribution that was consistent with transport of albumin from blood to cerebrospinal fluid. In adult plexus this pattern of immunostaining was absent. We propose a model of the cellular mechanism in which SPARC and GYPA, together with identified vesicle-associated membrane proteins (VAMPs) may act as receptors/transporters in developmentally regulated transfer of plasma proteins at the blood–CSF interface.

A cell's “redox” (oxidation and reduction) state is determined by the sum of all redox processes yielding reactive oxygen species (ROS), reactive nitrogen species (RNS), and other reactive intermediates. Low amounts of ROS/RNS are generated by different mechanisms in every cell and are important regulatory mediators in many signaling processes (redox signaling). When the physiological balance between the generation and elimination of ROS/RNS is disrupted, oxidative/nitrosative stress with persistent oxidative damage of the organism occurs. Oxidative stress has been suggested to act as initiator and/or mediator of many human diseases. The cerebral vasculature is particularly susceptible to oxidative stress, which is critical since cerebral endothelial cells play a major role in the creation and maintenance of the blood–brain barrier (BBB). This article will only contain a focused introduction on the biochemical background of redox signaling, since this has been reported already in a series of excellent recent reviews. The goal of this work is to increase the understanding of basic mechanisms underlying ROS/RNS-induced BBB disruption, with a focus on the role of matrix metalloproteinases, which, after all, appear to be a key mediator in the initiation and progression of BBB damage elicited by oxidative stress. Antioxid. Redox Signal. 15, 1305–1323.

Background Rotator cuff tears are a common cause of shoulder pain and impairment. Subacromial glucocorticoid injections are widely used for treatment of epiphenomenons of chronic impingement syndrome with the possible side effects of tendon rupture and impaired tendon healing. Methods Using qRT-PCR, western blot, immunoflourescence, and measurement of 3H-thymidine uptake we investigated the effects of the crystalline glucocorticoid triamcinolone acetonide (TAA) when added to the culture medium of isolated human rotator cuff tendon cells. Results After 2 weeks of incubation, the cells had lost their fibroblastic appearance and parallel orientation, which is characteristic of cellular degeneration in vivo. Moreover, expression and secretion of collagen I was strongly reduced, and there was a decrease in proliferation rate. Cell migration was blocked and the rate of expression of the matrix metalloproteinases MMP2, MMP8, MMP9, and MMP13 was reduced, but expression of TIMP1 (a tissue inhibitor of MMPs) was upregulated, indicating a reduction in the cellular capacity for tendon repair. In addition, changes in cellular differentiation were observed: the number of adipocytes increased and levels of the protein Sox9—a marker of differentiating and mature chondrocytes—were elevated in triamcinolone acetonide treated cells. Interpretation These results may indicate that the use of TAA is one reason for weaker mechanical tendon properties and for the high rate of re-rupture after supraspinatus tendon repair.

To investigate the role of aquaporin-mediated water transport during pollen grain germination and tube growth, Arabidopsis thaliana plasma membrane intrinsic proteins (PIPs) were expressed in pollen of Lilium longiflorum (lily). Successful expression of AtPIPs in particle-bombarded lily pollen grains was monitored by co-expression with fluorescent proteins and single-cell RT-PCR, and by measuring the water permeability coefficient (P(os)) in swelling assays using protoplasts prepared from transformed pollen grains and tubes. Expression of AtPIP1;1 and AtPIP1;2 in pollen grains resulted in P(os) values similar to those measured in nontransformed pollen grain protoplasts (6.65 +/- 2.41 microm s(-1)), whereas expression of AtPIP2 significantly increased P(os) (AtPIP2;1, 13.79 +/- 6.38; AtPIP2;2, 10.16 +/- 3.30 microm s(-1)). Transformation with combinations of AtPIP1 and AtPIP2 did not further enhance P(os). Native pollen tube protoplasts showed higher P(os) values (13.23 +/- 4.14 microm s(-1)) than pollen grain protoplasts but expression of AtPIP2;1 (18.85 +/- 7.60 microm s(-1)) did not significantly increase their P(os) values. Expression of none of the tested PIPs had any effect on pollen tube growth rates. The ectopic expression of AtPIP2s in lily pollen increased the water permeability of the plasma membrane in pollen grains, but not in pollen tubes. The measured endogenous water permeability does not limit water uptake during tube growth, but has to be regulated to prevent tube bursting.

Combining the patch-clamp method with single-cell reverse transcription polymerase chain reaction (scRT-PCR) a fusicoccin-induced current reflecting the activity of the plasma membrane H(+) ATPase of lily pollen protoplasts was measured and subsequently, the ATPase-encoding mRNAs were collected and amplified. Southern blot signals were observed in all 'patch-catch' experiments and could be detected even in 2560-fold dilutions of the pollen contents. H(+) ATPase mRNAs were detectable only in the vegetative but not in the generative cell of pollen as confirmed by immunolocalisation. In 15% of the scRT-PCR experiments, a random non-reproducibility of the PCR was observed, probably caused by varying amounts of ATPase mRNAs in the protoplasts.

Introduction Tissue barriers function as “gate keepers” between different compartments (usually blood and tissue) and are created by specialised membrane-associated proteins, located at the lateral plasma membrane of epithelial and endothelial cells. By sealing the paracellular space, such barriers impede the free diffusion of solutes and molecules across epithelial and endothelial monolayers, thereby creating an organ-specific homeostatic milieu. Tendon cells originate from yet poorly described precursor cells and develop in a particular “niche” close to vascular walls. Degenerative processes, trauma and injury severely disturb the internal milieu of the niche. Concomitantly, pathological alterations including hyperproliferation, erroneous tendon cell differentiation, and calcification take place [Astrom, 1995; Oliva, 2012]. It is a commonly held belief that the vasculature plays a crucial role in creating and maintaining a favourable milieu for tendon cells to develop, however, experimental evidence of how this is accomplished has been lacking. We now report that tendon cells are protected from the systemic circulation by a novel barrier, the blood-tendon barrier (BTB), located in the vascular wall of tendon vessels. The diffusion restraint exerted by the BTB is capable of defining and controlling the molecular composition of the tendon-specific niche, which after all governs proper tendon cell differentiation [Bi, 2007]. Methods RT-PCR, immunohistochemistry, transmission electron microscopy and in vivo tracer perfusion were performed to identify the expression of barrier-associated proteins and to demonstrate the “tightness” of tendon vessels in human Palmaris longus and mouse Achilles tendons. Results Perfusion with defined tracer substances revealed that the blood-tendon-barrier impedes the passive transport of macromolecules (≥ 10 kDa) from the blood stream to the surrounding tendon tissue (Figure 1), but is permeable to molecules Discussion The mechanisms and factors maintaining the internal milieu of the developmental niche in healthy tendons are not yet fully understood. Here, we describe a novel vascular structure, the blood-tendon barrer (BTB), which separates tendon tissue from the systemic circulation in intact human and murine tendons. The role of this structure in tendinopathy, tendon regeneration and tendon development remains to be elucidated. References Astrom, et al . Clin Orthop Relat Res. 1995;316:151–164, 1995 Bi, et al . Nat Med . 2007;13(10):1219–1227 Oliva, et al . BMC Med . 2012;10:95

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.

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.