ABSTRACT
In the cardiac microenvironment, cardiomyocytes (CMs) are embedded in an aligned and structured extracellular matrix (ECM) to maintain the coordinated contractile function of the heart. The cardiac fibroblast (cFB) is the main cell type responsible for producing and remodeling this matrix. In cardiac diseases, however, adverse remodeling and CM death may lead to deterioration of the aligned myocardial structure. Here, we present an in vitro cardiac model system with uniaxial and biaxial constraints to induce (an)isotropy in 3D microtissues, thereby mimicking 'healthy' aligned and 'diseased' disorganized cardiac matrices. A mixture of neonatal mouse CMs and cFBs was resuspended in a collagen-matrigel hydrogel and seeded to form microtissues to recapitulate the in vivo cellular composition. Matrix disarray led to a stellate cell shape and a disorganized sarcomere organization, while CMs in aligned matrices were more elongated and had aligned sarcomeres. Although matrix disarray has no detrimental effect on the force generated by the CMs, it did have a negative effect on the homogeneity of contraction force distribution. Furthermore, proliferation of the cFBs affected microtissue contraction as indicated by the negative correlation between the percentage of cFBs in the microtissues and their beating frequency. These results suggest that in regeneration of the diseased heart, reorganization of the disorganized matrix will contribute to recover the coordinated contraction but restoring the ratio in cellular composition (CMs and cFBs) is also a prerequisite to completely regain tissue function.
Subject(s)
Extracellular Matrix/physiology , Myocardial Contraction/physiology , Myocardium/cytology , Myocytes, Cardiac/physiology , Tissue Engineering/methods , Animals , Animals, Newborn , Anisotropy , Extracellular Matrix/ultrastructure , Finite Element Analysis , Mice , Mice, Inbred C57BL , Microscopy, Confocal , Microscopy, Fluorescence , Myocardium/ultrastructure , Myocytes, Cardiac/cytologyABSTRACT
Tumor cell plasticity enables certain types of highly malignant tumor cells to dedifferentiate and engage a plastic multipotent embryonic-like phenotype, which enables them to 'adapt' during tumor progression and escape conventional therapeutic strategies. This plastic phenotype of aggressive cancer cells enables them to express endothelial cell-specific markers and form tube-like structures, a phenotype that has been linked to aggressive behavior and poor prognosis. We demonstrate here that the transforming growth factor (TGF)-ß co-receptor endoglin, an endothelial cell marker, is expressed by tumor cells and its expression correlates with tumor cell plasticity in two types of human cancer, Ewing sarcoma and melanoma. Moreover, endoglin expression was significantly associated with worse survival of Ewing sarcoma patients. Endoglin knockdown in tumor cells interferes with tumor cell plasticity and reduces invasiveness and anchorage-independent growth in vitro. Ewing sarcoma and melanoma cells with reduced endoglin levels showed reduced tumor growth in vivo. Mechanistically, we provide evidence that endoglin, while interfering with TGF-ß signaling, is required for efficient bone morphogenetic protein, integrin, focal adhesion kinase and phosphoinositide-3-kinase signaling in order to maintain tumor cell plasticity. The present study delineates an important role of endoglin in tumor cell plasticity and progression of aggressive tumors.
Subject(s)
Antigens, CD/physiology , Melanoma/pathology , Receptors, Cell Surface/physiology , Sarcoma, Ewing/pathology , Animals , Antigens, CD/genetics , Base Sequence , Bone Morphogenetic Proteins/metabolism , Cell Division/physiology , Cell Line, Tumor , DNA Primers , Endoglin , Female , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Gene Knockdown Techniques , Humans , Immunohistochemistry , Mice , Mice, Nude , Neoplasm Invasiveness , Phosphatidylinositol 3-Kinases/metabolism , Receptors, Cell Surface/genetics , Signal Transduction , Transforming Growth Factor beta/metabolismABSTRACT
In skeletal muscle tissue engineering, it remains a challenge to produce mature, functional muscle tissue. Mimicking the in vivo niche in in vitro culture might overcome this problem. Niche components include, for example, extracellular matrix proteins, neighbouring cells, growth factors and physical factors such as the elasticity of the matrix. Previously, we showed the effects of matrix stiffness and protein coating on proliferation and differentiation of muscle progenitor cells in a two-dimensional (2D) situation. In the present study we have investigated the additional effect of electrical stimulation. More precisely, we investigated the effect of electrical stimulation on primary myoblast maturation when cultured on top of Matrigel™ - or laminin-coated substrates with varying elasticities. The effect of electrical stimulation on differentiation and maturation was found to be dependent on coating and stiffness. Although electrical stimulation enhanced myoblast maturation, the effect was mild. We therefore conclude that, with the current regimen, electrical stimulation is not essential to create functional, mature muscle tissue.
Subject(s)
Cell Differentiation , Elasticity , Extracellular Matrix Proteins/metabolism , Extracellular Matrix/metabolism , Muscle Fibers, Skeletal/cytology , Actinin/metabolism , Animals , Cells, Cultured , Electric Stimulation , Gene Expression Regulation , Male , Mice , Mice, Inbred C57BL , Muscle Contraction/physiology , Muscle Development/genetics , Muscle Fibers, Skeletal/metabolism , Muscle Proteins/genetics , Muscle Proteins/metabolism , Polymerase Chain Reaction , Time FactorsABSTRACT
The use of muscle progenitor cells (MPCs) for regenerative medicine has been severely compromised by their decreased proliferative and differentiative capacity after being cultured in vitro. We hypothesized the loss of pivotal niche factors to be the cause. Therefore, we investigated the proliferative and differentiative response of passage 0 murine MPCs to varying substrate elasticities and protein coatings and found that proliferation was influenced only by elasticity, whereas differentiation was influenced by both elasticity and protein coating. A stiffness of 21 kPa optimally increased the proliferation of MPCs. Regarding differentiation, we demonstrated that fusion of MPCs into myotubes takes place regardless of elasticity. However, ongoing maturation with cross-striations and contractions occurred only on elasticities higher than 3 kPa. Furthermore, maturation was fastest on poly-d-lysine and laminin coatings.
Subject(s)
Basement Membrane/metabolism , Cell Differentiation , Cell Proliferation , Muscle Development , Muscle Fibers, Skeletal/metabolism , Satellite Cells, Skeletal Muscle/metabolism , Signal Transduction , Animals , Basement Membrane/chemistry , Cells, Cultured , Collagen/metabolism , Collagen Type IV/metabolism , Drug Combinations , Elasticity , Laminin/metabolism , Male , Mice , Mice, Inbred C57BL , Polylysine/metabolism , Proteoglycans/metabolism , Time FactorsABSTRACT
The purpose of this study was to evaluate the effects of anginex on tumour angiogenesis assessed by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) on a clinical 1.5 Tesla MR system and with the clinically available contrast agent gadopentetate dimeglumine. C57BL/6 mice carrying B16F10 melanomas were treated with anginex, TNP-470 or saline. Tumour growth curves and microvessel density (MVD) were recorded to establish the effects of treatment. DCE-MRI was performed on day 16 after tumour inoculation, and the endothelial transfer coefficients of the microvessel permeability surface-area product (K(PS)) were calculated using a two-compartment model. Both anginex and TNP-470 resulted in smaller tumour volumes (P<0.0001) and lower MVD (P <0.05) compared to saline. Treatment with anginex resulted in a 64% reduction (P<0.01) of tumour K(PS) and TNP-470 resulted in a 44% reduction (P=0.17), compared to saline. DCE-MRI with a clinically available, small-molecular contrast agent can therefore be used to evaluate the angiostatic effects of anginex and TNP-470 on tumour angiogenesis.
