RESUMO
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.
Assuntos
Tendão do Calcâneo/metabolismo , Transdução de Sinais/fisiologia , Fator D de Crescimento do Endotélio Vascular/metabolismo , Animais , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Matriz Extracelular/metabolismo , Feminino , Humanos , Masculino , Camundongos , Neovascularização Patológica/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos Lew , Ratos Sprague-Dawley , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Human platelet lysate (HPL) is an efficient alternative for animal serum supplements, significantly enhancing stromal cell proliferation. However, the molecular mechanism behind this growth-promoting effect remains elusive. The aim of this study was to investigate the effect of HPL on cell cycle gene expression in different human stromal cells and to identify the main key players that mediate HPL's growth-enhancing effect. RT-qPCR and an antibody array revealed significant upregulation of cell cycle genes in stromal cells cultured in HPL. As HPL is rich in growth factors that are ligands of tyrosine kinase receptor (TKR) pathways, we used TKR inhibitors and could significantly reduce cell proliferation. Genome profiling, RT-qPCR and Western blotting revealed an enhanced expression of the transcription factors signal transducer and activator of transcription 3 (STAT3) and MYC, both known TKR downstream effectors and stimulators of cell proliferation, in response to HPL. In addition, specifically blocking STAT3 resulted in reduced cell proliferation and expression of cell cycle genes. Our data indicate that HPL-enhanced cell proliferation can, at least in part, be explained by the TKR-enhanced expression of STAT3 and MYC, which in turn induce the expression of genes being involved in the promotion and control of the cell cycle.
Assuntos
Células-Tronco Mesenquimais , Proteínas Proto-Oncogênicas c-myc , Fator de Transcrição STAT3 , Animais , Humanos , Plaquetas/metabolismo , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Células-Tronco Mesenquimais/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Células Estromais/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
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.
Assuntos
Bevacizumab/uso terapêutico , Traumatismos dos Tendões/tratamento farmacológico , Tendão do Calcâneo/patologia , Animais , Bevacizumab/farmacologia , Modelos Animais de Doenças , Módulo de Elasticidade , Feminino , Marcha/fisiologia , Neovascularização Fisiológica/efeitos dos fármacos , Ratos , Ratos Endogâmicos Lew , Traumatismos dos Tendões/patologia , Resistência à Tração , Cicatrização/efeitos dos fármacosRESUMO
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.
Assuntos
Tendão do Calcâneo , Hipersensibilidade , Tendinopatia , Tendão do Calcâneo/patologia , Animais , Humanos , Hipersensibilidade/complicações , Hipersensibilidade/patologia , Inflamação/patologia , Metaloproteinase 2 da Matriz , Camundongos , Tendinopatia/etiologia , Tendinopatia/patologiaRESUMO
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.
Assuntos
Predisposição Genética para Doença , Osteonectina , Tendões/fisiologia , Animais , Homeostase , Humanos , Ligamentos , Camundongos , Camundongos Knockout , Osteonectina/genéticaRESUMO
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.
Assuntos
Lesões do Manguito Rotador/etiologia , Manguito Rotador/patologia , Tendões/patologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Feminino , Humanos , Masculino , Metaloproteinase 2 da Matriz/genética , Pessoa de Meia-Idade , Estudos Prospectivos , Fatores de Risco , Adulto JovemRESUMO
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.
Assuntos
MicroRNAs/sangue , Lesões do Manguito Rotador/diagnóstico , Idoso , Biópsia , Estudos de Casos e Controles , Feminino , Humanos , Masculino , MicroRNAs/análise , MicroRNAs/fisiologia , Pessoa de Meia-Idade , Lesões do Manguito Rotador/etiologia , Tendões/química , Tendões/patologiaRESUMO
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.
Assuntos
Tendão do Calcâneo/citologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Organoides/citologia , Células-Tronco/citologia , Engenharia Tecidual/métodos , Tendão do Calcâneo/crescimento & desenvolvimento , Tendão do Calcâneo/metabolismo , Animais , Células Cultivadas , Colágeno/química , Organoides/crescimento & desenvolvimento , Ratos , Células-Tronco/fisiologia , Alicerces Teciduais/químicaRESUMO
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.
Assuntos
Cicatriz/metabolismo , Neovascularização Patológica/metabolismo , Animais , Cicatriz/tratamento farmacológico , Cicatriz/patologia , Sistemas de Liberação de Medicamentos , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Cicatrização/efeitos dos fármacosRESUMO
Tendinopathy is accompanied by a cascade of inflammatory events promoting tendon degeneration. Among various cytokines, interleukin-1ß 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-1ß-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-1ß 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.
Assuntos
Tendão do Calcâneo , Magnetoterapia/métodos , Tendinopatia/terapia , Tendão do Calcâneo/citologia , Tendão do Calcâneo/imunologia , Animais , Apoptose/imunologia , Interleucina-1beta/imunologia , Ratos , Ratos Endogâmicos F344 , Receptores Tipo II de Interleucina-1/imunologiaRESUMO
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-1ß, 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.
Assuntos
Receptor 1 de Quimiocina CX3C/metabolismo , Quimiocina CX3CL1/metabolismo , Macrófagos/metabolismo , Tendões/citologia , Animais , Movimento Celular , Epirregulina/metabolismo , Feminino , Proteínas de Fluorescência Verde/metabolismo , Humanos , Sistema Imunitário , Inflamação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ratos , Ratos Endogâmicos F344RESUMO
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.
Assuntos
Aquaporinas/fisiologia , Proteínas de Arabidopsis/fisiologia , Permeabilidade da Membrana Celular/fisiologia , Lilium/fisiologia , Tubo Polínico/fisiologia , Pólen/fisiologia , Aquaporinas/genética , Proteínas de Arabidopsis/genética , Fluorescência , Expressão Gênica , Tubo Polínico/crescimento & desenvolvimento , Protoplastos/fisiologia , Transformação Genética , Água/fisiologiaRESUMO
â¢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.
RESUMO
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.
Assuntos
Anticorpos Monoclonais/biossíntese , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/imunologia , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/imunologia , Sequência de Aminoácidos , Animais , Arabidopsis/genética , Feminino , Vetores Genéticos , Hibridomas/imunologia , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Plantas Geneticamente Modificadas , Plasmídeos/genética , Coelhos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Saccharomyces cerevisiae/genética , Nicotiana/genética , Vacinas de DNA/genéticaRESUMO
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.
Assuntos
Células da Medula Óssea/metabolismo , Diferenciação Celular , Fêmur/lesões , Fêmur/metabolismo , Osteogênese , Tendões/metabolismo , Animais , Células da Medula Óssea/patologia , Fêmur/patologia , Masculino , Ratos , Ratos Endogâmicos F344 , Células Estromais/metabolismo , Células Estromais/patologia , Tendões/patologiaRESUMO
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.
Assuntos
Tendão do Calcâneo/metabolismo , Dieta , Glucose , Traumatismos dos Tendões/metabolismo , Cicatrização , Animais , Fenômenos Biomecânicos , Proliferação de Células , Marcha , Expressão Gênica , Tamanho do Órgão , Ratos , Traumatismos dos Tendões/patologiaRESUMO
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.
Assuntos
Envelhecimento/metabolismo , Pleiotropia Genética , Osteonectina/metabolismo , Tendões/crescimento & desenvolvimento , Tendões/metabolismo , Adipogenia , Animais , Biomarcadores/metabolismo , Fenômenos Biomecânicos , Forma Celular , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Metabolismo dos Lipídeos , Masculino , Camundongos Endogâmicos C57BL , Osteonectina/deficiência , Ratos , Células-Tronco/citologia , Tendões/fisiologia , Tendões/ultraestruturaRESUMO
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.
Assuntos
Pólen/enzimologia , Bombas de Próton/isolamento & purificação , ATPases Translocadoras de Prótons/isolamento & purificação , RNA Mensageiro/isolamento & purificação , Lilium , Técnicas de Patch-Clamp , Pólen/genética , Bombas de Próton/genética , Bombas de Próton/metabolismo , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Protoplastos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
TaqMan(R) proximity ligation technology (TaqMan(R) PLA) is an innovative advancement of immuno PCR. It allows a fast and quantitative detection of vicinal proteins or protein-protein interactions from cell lysates by combining antibody-antigen binding with a real-time PCR detection. We tested if TaqMan(R) PLA also was applicable to investigate and relatively quantitate adhesion receptor heterodimers such as the alpha-4/beta-1 integrin on the surface of intact cells. Both, alpha-4, beta-1 and the alpha-4/beta-1 heterodimer were detected on the surface of lymphocytes by TaqMan(R) PLA. Results were specific, reproducible and comparable to flow cytometric data. However, preciseness of reactions varied dependent on the antibody pairs used. Co-detection of proximate identical subunits suggested clusters of alpha-4 and/or beta-1 on the cell surface which we confirmed by microscopy. We conclude that real-time PCR-based TaqMan(R) PLA is of limited applicability to investigate heterodimeric receptor molecules such as the alpha-4/beta-1 integrin. Determination of an abundance ratio of alpha-4/beta-1 in relation to total alpha-4 or beta-1 was not possible and real-time detection did not allow conclusions on the surface distribution of molecules. The related in situ PLA developed for microscopy allows visualizing proximate protein interactions and might be an interesting alternative for research into receptor heterodimers and their surface distribution on immune cells.
Assuntos
Integrina alfa4/análise , Integrina beta1/análise , Complexo Glicoproteico GPIb-IX de Plaquetas/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Linfócitos T/citologia , Anticorpos/química , Reações Antígeno-Anticorpo , Adesão Celular , Citometria de Fluxo , Corantes Fluorescentes , Humanos , Integrina alfa4/imunologia , Integrina beta1/imunologia , Sondas de Oligonucleotídeos/química , Complexo Glicoproteico GPIb-IX de Plaquetas/imunologia , Cultura Primária de Células , Multimerização Proteica , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Linfócitos T/imunologiaRESUMO
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 blood-CSF 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 blood-CSF 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.