RESUMO
Notch receptors control tissue morphogenic processes that involve coordinated changes in cell architecture and gene expression, but how a single receptor can produce these diverse biological outputs is unclear. Here, we employ a 3D model of a human ductal epithelium to reveal tissue morphogenic defects result from loss of Notch1, but not Notch1 transcriptional signaling. Instead, defects in duct morphogenesis are driven by dysregulated epithelial cell architecture and mitogenic signaling which result from the loss of a transcription-independent, Notch1 cortical signaling mechanism that ultimately functions to stabilize adherens junctions and cortical actin. We identify that Notch1 localization and cortical signaling are tied to apical-basal cell restructuring and discover that a Notch1-FAM83H interaction underlies control of epithelial adherens junctions and cortical actin. Together, these results offer new insights into Notch1 signaling and regulation and advance a paradigm in which transcriptional and cell adhesive programs might be coordinated by a single receptor.
Assuntos
Actinas , Junções Aderentes , Adesão Celular , Receptor Notch1 , Humanos , Junções Aderentes/genética , Proliferação de Células , Células Epiteliais , Proteínas , Receptor Notch1/genética , Transdução de SinaisRESUMO
Intratumor heterogeneity associates with poor patient outcome. Stromal stiffening also accompanies cancer. Whether cancers demonstrate stiffness heterogeneity, and if this is linked to tumor cell heterogeneity remains unclear. We developed a method to measure the stiffness heterogeneity in human breast tumors that quantifies the stromal stiffness each cell experiences and permits visual registration with biomarkers of tumor progression. We present Spatially Transformed Inferential Force Map (STIFMap) which exploits computer vision to precisely automate atomic force microscopy (AFM) indentation combined with a trained convolutional neural network to predict stromal elasticity with micron-resolution using collagen morphological features and ground truth AFM data. We registered high-elasticity regions within human breast tumors colocalizing with markers of mechanical activation and an epithelial-to-mesenchymal transition (EMT). The findings highlight the utility of STIFMap to assess mechanical heterogeneity of human tumors across length scales from single cells to whole tissues and implicates stromal stiffness in tumor cell heterogeneity.
Assuntos
Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/patologia , Fenômenos Mecânicos , Elasticidade , Colágeno , Redes Neurais de Computação , Microscopia de Força Atômica/métodosRESUMO
Tumor metastasis via the circulation requires crossing the vascular barrier twice: first, during intravasation when tumor cells disseminate from the primary site through proximal vasculature, and second, during extravasation, when tumor cells exit the circulation to form distant metastatic seeds. During these key metastatic events, chemomechanical signaling between tumor cells and endothelial cells elicits reciprocal changes in cell morphology and behavior that are necessary to breach the vessel wall. Existing experimental systems have provided a limited understanding of the diverse mechanisms underlying tumor-endothelial interactions during intravasation and extravasation. Recent advances in microphysiological systems have revolutionized the ability to generate miniaturized human tissues with tailored three-dimensional architectures, physiological cell interfaces, and precise chemical and physical microenvironments. By doing so, microphysiological systems enable experimental access to complex morphogenic processes associated with human tumor progression with unprecedented resolution and biological control. Here, we discuss recent examples in which microphysiological systems have been leveraged to reveal new mechanistic insight into cellular and molecular control systems operating at the tumor-endothelial interface during intravasation and extravasation.
Assuntos
Células Endoteliais , Neoplasias , Humanos , Células Endoteliais/patologia , Neoplasias/patologia , Endotélio , Transdução de Sinais , Metástase Neoplásica , Microambiente TumoralAssuntos
Velocidade do Fluxo Sanguíneo/fisiologia , Membrana Eritrocítica/fisiologia , Angiofluoresceinografia , Fluxo Sanguíneo Regional/fisiologia , Esclera/irrigação sanguínea , Idoso de 80 Anos ou mais , Corantes/administração & dosagem , Feminino , Humanos , Verde de Indocianina/administração & dosagem , Masculino , Pessoa de Meia-IdadeRESUMO
Changes in retinal blood flow may be involved in the pathogenesis of glaucoma and other ocular diseases. Erythrocyte mediated velocimetry (EMV) is a novel technique where indocyanine green (ICG) dye is sequestered in erythrocyte ghosts and autologously re-injected to allow direct visualization of erythrocytes for in vivo measurement of speed. The purpose of this study is to determine the mean erythrocyte speed in the retinal microvasculature, as well as the intravisit and intervisit variability of EMV. Data from 23 EMV sessions from control, glaucoma suspect, and glaucoma patients were included in this study. In arteries with an average diameter of 43.11 µm ± 6.62 µm, the mean speed was 7.17 mm/s ± 2.35 mm/s. In veins with an average diameter of 45.87 µm ± 12.04 µm, the mean speed was 6.05 mm/s ± 1.96 mm/s. Intravisit variability, as measured by the mean coefficient of variation, was 3.57% (range 0.44-9.68%). Intervisit variability was 4.85% (range 0.15-8.43%). EMV may represent reliable method for determination of retinal blood speed, potentially allowing insights into the effects of pharmacologic agents or pathogenesis of ocular diseases.
Assuntos
Velocidade do Fluxo Sanguíneo/fisiologia , Eritrócitos/fisiologia , Glaucoma/fisiopatologia , Microvasos/fisiopatologia , Vasos Retinianos/fisiopatologia , Idoso , Estudos de Casos e Controles , Feminino , Humanos , Pressão Intraocular , Masculino , Pessoa de Meia-Idade , ReologiaRESUMO
Retinal blood flow is an emerging biomarker in ocular and systemic disease. Erythrocyte mediated angiography (EMA) is a novel technique that provides an easily interpretable blood flow velocity quantification by directly tracing individual moving erythrocyte ghosts over time in vivo, imaged using a scanning laser ophthalmoscope (Heidelberg Retina Angiograph platform). This tracking procedure, however, requires time-consuming manual analysis to determine blood flow. To overcome this current bottleneck, we developed an objective and automated velocimetry approach, EMA - Automated Velocimetry (EMA-AV), which is based on a modified sequential Monte Carlo method. The intra-class correlation coefficient (ICC) between trained human graders and EMA-AV is 0.98 for mean vessel velocity estimation and 0.92 for frame by frame erythrocyte velocity estimation. This study proves EMA-AV is a reliable tool for quantification of retinal microvascular velocity and flow and establishes EMA-AV as a reliable and interpretable tool for quantifying retinal microvascular velocity.
RESUMO
BACKGROUND: Blood-brain barrier dysfunction is associated with many late-stage neurodegenerative diseases. An emerging question is whether the mutations associated with neurodegenerative diseases can independently lead to blood-brain barrier (BBB) dysfunction. Studies from patient-derived induced pluripotent stem cells suggest that mutations associated with neurodegenerative disease are non-cell autonomous, resulting in gain of toxic function in derived neurons and astrocytes. Here we assess whether selected mutations associated with neurodegenerative diseases can contribute to impairment of the blood-brain barrier. METHODS: We assessed barrier function of confluent monolayers of human brain microvascular endothelial cells (hBMECs) derived from induced pluripotent stem cells (iPSC) from three healthy individuals and eight individuals with neurodegenerative disease. We systematically assessed protein and gene expression of BBB biomarkers, transendothelial resistance (TEER), permeability of Lucifer yellow, permeability of D-glucose, permeability of rhodamine 123, the efflux ratio of rhodamine 123, and P-gp inhibition using Tariquidar for confluent monolayers of human brain microvascular endothelial cell (hBMECs). RESULTS: We provide evidence supporting the hypothesis that mutations associated with neurodegenerative disease can independently cause BBB dysfunction. These functional changes are not catastrophic since barrier breakdown would result in BBB impairment during development. Synergistic interactions between non-cell autonomous cerebrovascular dysfunction and the effects of gain-of-toxic function in neurons (e.g. toxic oligomers) are likely to increase disease burden through a positive feedback mechanism. CONCLUSIONS: These results suggest that the accumulation of defects in brain microvascular endothelial cells may ultimately lead to impairment of the BBB. Small changes in barrier function over time could lead to accumulated defects that result in positive feedback to unrelated central nervous system diseases.
Assuntos
Barreira Hematoencefálica/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Mutação/fisiologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Adulto , Idoso , Barreira Hematoencefálica/patologia , Células Endoteliais/patologia , Células Endoteliais/fisiologia , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Masculino , Pessoa de Meia-Idade , Doenças Neurodegenerativas/patologiaRESUMO
The high rate of drug attrition caused by cardiotoxicity is a major challenge for drug development. Here, we developed a reflective lens-free imaging (RLFI) approach to non-invasively record in vitro cell deformation in cardiac monolayers with high temporal (169 fps) and non-reconstructed spatial resolution (352 µm) over a field-of-view of maximally 57 mm2. The method is compatible with opaque surfaces and silicon-based devices. Further, we demonstrated that the system can detect the impairment of both contractility and fast excitation waves in cardiac monolayers. Additionally, the RLFI device was implemented on a CMOS-based microelectrode array to retrieve multi-parametric information of cardiac cells, thereby offering more in-depth analysis of drug-induced (cardiomyopathic) effects for preclinical cardiotoxicity screening applications.
RESUMO
BACKGROUND: Transwell-based models of the blood-brain barrier (BBB) incorporating monolayers of human brain microvascular endothelial cells (dhBMECs) derived from induced pluripotent stem cells show many of the key features of the BBB, including expression of transporters and efflux pumps, expression of tight junction proteins, and physiological values of transendothelial electrical resistance. The fabrication of 3D BBB models using dhBMECs has so far been unsuccessful due to the poor adhesion and survival of these cells on matrix materials commonly used in tissue engineering. METHODS: To address this issue, we systematically screened a wide range of matrix materials (collagen I, hyaluronic acid, and fibrin), compositions (laminin/entactin), protein coatings (fibronectin, laminin, collagen IV, perlecan, and agrin), and soluble factors (ROCK inhibitor and cyclic adenosine monophosphate) in 2D culture to assess cell adhesion, spreading, and barrier function. RESULTS: Cell coverage increased with stiffness of collagen I gels coated with collagen IV and fibronectin. On 7 mg mL-1 collagen I gels coated with basement membrane proteins (fibronectin, collagen IV, and laminin), cell coverage was high but did not reliably reach confluence. The transendothelial electrical resistance (TEER) on collagen I gels coated with basement membrane proteins was lower than on coated transwell membranes. Agrin, a heparin sulfate proteoglycan found in basement membranes of the brain, promoted monolayer formation but resulted in a significant decrease in transendothelial electrical resistance (TEER). However, the addition of ROCK inhibitor, cAMP, or cross-linking the gels to increase stiffness, resulted in a significant improvement of TEER values and enabled the formation of confluent monolayers. CONCLUSIONS: Having identified matrix compositions that promote monolayer formation and barrier function, we successfully fabricated dhBMEC microvessels in cross-linked collagen I gels coated with fibronectin and collagen IV, and treated with ROCK inhibitor and cAMP. We measured apparent permeability values for Lucifer yellow, comparable to values obtained in the transwell assay. During these experiments we observed no focal leaks, suggesting the formation of tight junctions that effectively block paracellular transport.