scholarly journals Microvascular Sprouting, Extension, and Creation of New Capillary Connections with Adaptation of the Neighboring Astrocytes in Adult Mouse Cortex under Chronic Hypoxia

2013 ◽  
Vol 34 (2) ◽  
pp. 325-331 ◽  
Author(s):  
Kazuto Masamoto ◽  
Hiroyuki Takuwa ◽  
Chie Seki ◽  
Junko Taniguchi ◽  
Yoshiaki Itoh ◽  
...  
Keyword(s):  
Adult Mouse ◽  
Fluorescent Protein ◽  
Vascular Endothelial Cells ◽  
Three Dimensional ◽  
Vascular Endothelial ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Mouse Cortex ◽  
Hypoxia Adaptation ◽  
Green Fluorescent

The present study aimed to determine the spatiotemporal dynamics of microvascular and astrocytic adaptation during hypoxia-induced cerebral angiogenesis. Adult C57BL/6J and Tie2-green fluorescent protein (GFP) mice with vascular endothelial cells expressing GFP were exposed to normobaric hypoxia for 3 weeks, whereas the three-dimensional microvessels and astrocytes were imaged repeatedly using two-photon microscopy. After 7 to14 days of hypoxia, a vessel sprout appeared from the capillaries with a bump-like head shape (mean diameter 14  μm), and stagnant blood cells were seen inside the sprout. However, no detectable changes in the astrocyte morphology were observed for this early phase of the hypoxia adaptation. More than 50% of the sprouts emerged from capillaries 60  μm away from the center penetrating arteries, which indicates that the capillary distant from the penetrating arteries is a favored site for sprouting. After 14 to 21 days of hypoxia, the sprouting vessels created a new connection with an existing capillary. In this phase, the shape of the new vessel and its blood flow were normalized, and the outside of the vessels were wrapped with numerous processes from the neighboring astrocytes. The findings indicate that hypoxia-induced cerebral angiogenesis provokes the adaptation of neighboring astrocytes, which may stabilize the blood–brain barrier in immature vessels.

scholarly journals Intravital Confocal and Two-Photon Imaging of Dual-Color Cells and Extracellular Matrix Mimics

2013 ◽  
Vol 19 (1) ◽  
pp. 201-212 ◽  
Author(s):  
Ufuk Bal ◽  
Volker Andresen ◽  
Brenda Baggett ◽  
Urs Utzinger
Keyword(s):  
Extracellular Matrix ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Second Harmonic ◽  
Three Dimensional ◽  
Confocal Imaging ◽  
Collagen I ◽  
Two Photon ◽  
Significant Difference ◽  
Green Fluorescent

AbstractWe report our efforts in identifying optimal scanning laser microscope parameters to study cells in three-dimensional culture. For this purpose we studied contrast of extracellular matrix (ECM) mimics, as well as signal attenuation, and bleaching of red and green fluorescent protein labeled cells. Confocal backscattering, second harmonic generation (SHG), and autofluorescence were sources of contrast in ECM mimics. All common ECM mimics exhibit contrast observable with confocal reflectance microscopy. SHG imaging on collagen I based hydrogels provides high contrast and good optical penetration depth. Agarose is a useful embedding medium because it allows for large optical penetration and exhibits minimal autofluorescence. We labeled breast cancer cells' outline with DsRed2 and nucleus with enhanced green fluorescent protein (eGFP). We observed significant difference both for the bleaching rates of eGFP and DsRed2 where bleaching is strongest during two-photon excitation (TPE) and smallest during confocal imaging. But for eGFP the bleaching rate difference is smaller than for DsRed2. After a few hundred microns depth in a collagen I hydrogel, TPE fluorescence of DsRed2 becomes twice as strong compared to confocal imaging. In fibrin and agarose gels, the imaging depth will need to be beyond 1 mm to notice a TPE advantage.

scholarly journals Accelerated Fibrinolysis and Its Propagation on Vascular Endothelial Cells by Secreted and Retained tPA

2012 ◽  
Vol 2012 ◽  
pp. 1-5 ◽  
Author(s):  
Tetsumei Urano ◽  
Yuko Suzuki
Keyword(s):  
Endothelial Cells ◽  
Fluorescent Protein ◽  
Vascular Endothelial Cells ◽  
Tissue Type ◽  
Dependent Manner ◽  
Vascular Endothelial ◽  
Type Plasminogen Activator ◽  
Lysine Residues ◽  
Associated Proteins ◽  
Green Fluorescent

We successfully visualized the secretory dynamics of tissue-type plasminogen activator (tPA) tagged by green fluorescent protein (tPA-GFP) from cultured vascular endothelial cells (VECs) using total internal reflection fluorescence (TIRF) microscopy and demonstrated that tPA-GFP secreted from VECs was retained on cell surfaces in a heavy-chain-dependent manner. Progressive binding of Alexa568-labeled Glu-plasminogen was also observed on the surface of active tPA-GFP expressing cells via lysine binding sites (LBS), which was not observed on inactive mutant tPA-GFP expressing cells. These results suggest that retained tPA on VECs effectively activated plasminogen to plasmin, which then facilitated the binding of additional plasminogen on the cell surface by proteolytically cleaving surface-associated proteins and exposing their C-terminal lysine residues. Thus prolonged retention of tPA appeared to play an important role in initiating and amplifying plasmin generation on VECs. LBS-dependent binding of plasminogen was also observed as a narrow band at the lytic front of the fibrin mesh formed on active tPA-GFP expressing cells, which expanded outward as the lytic area increased. This binding was not observed on inactive mutant tPA-GFP expressing cells or in the presence of aprotinin. The binding of plasminogen to partially digested fibrin appears to be indispensable for spontaneous fibrinolysis.

scholarly journals Pleiotrophin produced by multiple myeloma induces transdifferentiation of monocytes into vascular endothelial cells: a novel mechanism of tumor-induced vasculogenesis

Blood ◽  
2009 ◽  
Vol 113 (9) ◽  
pp. 1992-2002 ◽  
Author(s):  
Haiming Chen ◽  
Richard A. Campbell ◽  
Yunchao Chang ◽  
Mingjie Li ◽  
Cathy S. Wang ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Fluorescent Protein ◽  
Vascular Endothelial Cells ◽  
Human Peripheral Blood ◽  
Vascular Endothelial Cell ◽  
Human Monocyte ◽  
Angiogenic Factor ◽  
Vascular Endothelial ◽  
Protein Markers ◽  
Green Fluorescent

Enhanced angiogenesis is a hallmark of cancer. Pleiotrophin (PTN) is an angiogenic factor that is produced by many different human cancers and stimulates tumor blood vessel formation when it is expressed in malignant cancer cells. Recent studies show that monocytes may give rise to vascular endothelium. In these studies, we show that PTN combined with macrophage colony-stimulating factor (M-CSF) induces expression of vascular endothelial cell (VEC) genes and proteins in human monocyte cell lines and monocytes from human peripheral blood (PB). Monocytes induce VEC gene expression and develop tube-like structures when they are exposed to serum or cultured with bone marrow (BM) from patients with multiple myeloma (MM) that express PTN, effects specifically blocked with antiPTN antibodies. When coinjected with human MM cells into severe combined immunodeficient (SCID) mice, green fluorescent protein (GFP)–marked human monocytes were found incorporated into tumor blood vessels and expressed human VEC protein markers and genes that were blocked by anti-PTN antibody. Our results suggest that vasculogenesis in human MM may develop from tumoral production of PTN, which orchestrates the transdifferentiation of monocytes into VECs.

Automatic Counting of Intra-Cellular Ribonucleo-Protein Aggregates in Saccharomyces cerevisiae Using a Textural Approach

2019 ◽  
Vol 25 (1) ◽  
pp. 164-179
Author(s):  
Ambroise Marin ◽  
Emmanuel Denimal ◽  
Lucie Bertheau ◽  
Stéphane Guyot ◽  
Ludovic Journaux ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fluorescent Protein ◽  
Protein Aggregates ◽  
Point Of View ◽  
Zernike Moment ◽  
Two Photon ◽  
Haralick Features ◽  
Green Fluorescent ◽  
Two Photon Fluorescence

AbstractIn the context of microbiology, recent studies show the importance of ribonucleo-protein aggregates (RNPs) for the understanding of mechanisms involved in cell responses to specific environmental conditions. The assembly and disassembly of aggregates is a dynamic process, the characterization of the stage of their evolution can be performed by the evaluation of their number. The aim of this study is to propose a method to automatically determine the count of RNPs. We show that the determination of a precise count is an issue by itself and hence, we propose three textural approaches: a classical point of view using Haralick features, a frequency point of view with generalized Fourier descriptors, and a structural point of view with Zernike moment descriptors (ZMD). These parameters are then used as inputs for a supervised classification in order to determine the most relevant. An experiment using a specific Saccharomyces cerevisiae strain presenting a fusion between a protein found in RNPs (PAB1) and the green fluorescent protein was performed to benchmark this approach. The fluorescence was observed with two-photon fluorescence microscopy. Results show that the textural approach, by mixing ZMD with Haralick features, allows for the characterization of the number of RNPs.

Interaction between vascular endothelial cells and vascular intimal spindle-shaped cells in vitro

1983 ◽  
Vol 60 (1) ◽  
pp. 89-102
Author(s):  
D de Bono ◽  
C. Green
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Three Dimensional ◽  
Vascular Endothelial ◽  
Pure Cultures ◽  
Species Specific ◽  
Endothelial Growth Factor

The interactions between human or bovine vascular endothelial cells and fibroblast-like vascular intimal spindle-shaped cells have been studied in vitro, using species-specific antibodies to identify the different components in mixed cultures. Pure cultures of endothelial cells grow as uniform, nonoverlapping monolayers, but this growth pattern is lost after the addition of spindle cells, probably because the extracellular matrix secreted by the latter causes the endothelial cells to modify the way they are attached to the substrate. The result is a network of tubular aggregates of endothelial cells in a three-dimensional ‘polylayer’ of spindle-shaped cells. On the other hand, endothelial cells added to growth-inhibited cultures of spindle-shaped cells will grow in sheets over the surface of the culture. Human endothelial cells grown in contact with spindle-shaped cells have a reduced requirement for a brain-derived endothelial growth factor. The interactions of endothelial cells and other connective tissue cells in vitro may be relevant to the mechanisms of endothelial growth and blood vessel formation in vivo, and emphasize the potential importance of extracellular matrix in controlling endothelial cell behaviour.

Developmental derivation of vocal fold mucosa from human induced pluripotent stem cells

2019 ◽  
Author(s):  
Vlasta Lungova ◽  
Susan Thibeault
Keyword(s):  
Epithelial Cells ◽  
Pluripotent Stem Cells ◽  
Vocal Fold ◽  
Fluorescent Protein ◽  
Three Dimensional ◽  
Stratified Squamous Epithelium ◽  
Three Dimensional Models ◽  
Induced Pluripotent ◽  
Green Fluorescent

Abstract Development of treatments for vocal dysphonia has been inhibited by lack of human vocal fold (VF) mucosa models because of difficulty in procuring VF epithelial cells, epithelial cells’ limited proliferative capacity and absence of cell lines. We report development of engineered VF mucosae from hiPSC, transfected via TALEN constructs for green fluorescent protein, that mimic development of VF epithelial cells in utero. Modulation of FGF signaling achieves stratified squamous epithelium from definitive and anterior foregut derived cultures. Robust culturing of these cells on collagen-fibroblast constructs produces three-dimensional models comparable to in vivo VF mucosa.

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