Abstract TP471: Oxygen-Glucose Deprivation and Denser Extracellular Matrix Cause Cerebral Capillary Endothelial Cells to Lose Elasticity

Stroke ◽  
2020 ◽  
Vol 51 (Suppl_1) ◽  
Author(s):  
Alexis Mack ◽  
Michael Maniskas ◽  
Caroline Reynolds ◽  
Akihiko Urayama
Keyword(s):  
Extracellular Matrix ◽  
Collagen Type ◽  
Acute Stress ◽  
Culture Media ◽  
Membrane Surface ◽  
Glucose Deprivation ◽  
Oxygen Glucose Deprivation ◽  
Collagen Type Iv ◽  
Type Iv ◽  
Energy Deprivation

Objective: Cerebral ischemia affects the mechano-response of vasculature and the remodeling of extracellular matrix (ECM) components. The present study investigates how oxygen-glucose deprivation (OGD) affects the brain endothelial cell (BEC) stiffness. We further aim to study whether ECM components affect the cellular stiffness in normal and energy deprived conditions. Methods: Primary human BECs received a 16h OGD followed by the reperfusion with normal culture media. Young’s Modulus (YM) and topography in the BEC surface were determined by atomic force microscopy (AFM). To test the effect of ECM composition on the BEC stiffness, various concentrations (5-500 uM) of collagen type IV were used for culture dish coating. BECs on the coating beds underwent 16h OGD and served for YM measurements. Per dish, 10-20 single BECs were measured. Results: BECs under OGD conditions became significantly stiffer (2.1-fold, *P<0.05) than the normoxic control, and the increased stiffness sustained even after the reperfusion for 24h, indicating energy deprivation is a primary factor for the increased stiffness under acute stress conditions. AFM topography confirmed that OGD induced rough and filamentous plasma membrane surface in BECs, suggesting a cytoskeletal change was induced by OGD. BECs grown on a 10x denser collagen type IV (500 uM) layer showed a significant increase (2.3-fold, ***P<0.001) in cellular stiffness, compared with the conventional concentration of 50 uM, suggesting that denser extracellular matrix makes BEC stiffer. Also, in BECs cultured on a denser ECM layer, OGD response was absent in terms of cellular stiffness. Conclusion: The present study found that OGD increased BEC stiffness. In addition, increasing the density of the ECM alone was enough to generate stiffer BECs. These mechanical properties of BECs may be a physical factor mediating endothelial responses after stroke.

scholarly journals Differential expression of parietal epithelial cell and podocyte extracellular matrix proteins in focal segmental glomerulosclerosis and diabetic nephropathy

2019 ◽  
Vol 317 (6) ◽  
pp. F1680-F1694 ◽  
Author(s):  
Gek Cher Chan ◽  
Diana G. Eng ◽  
Jeffrey H. Miner ◽  
Charles E. Alpers ◽  
Kelly Hudkins ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Diabetic Nephropathy ◽  
Epithelial Cell ◽  
Focal Segmental Glomerulosclerosis ◽  
Collagen Type ◽  
Collagen Type Iv ◽  
Type Iv ◽  
Ecm Proteins ◽  
Segmental Glomerulosclerosis ◽  
Parietal Epithelial Cell

In healthy glomeruli, parietal epithelial cell (PEC)-derived extracellular matrix (ECM) proteins include laminin-β1, perlecan, and collagen type IV-α2 and podocyte-specific ECM proteins include laminin-β2, agrin, and collagen type IV-α4. This study aimed to define individual ECM protein isoform expression by PECs in both experimental and human focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy (DN) and to determine if changes were CD44 dependent. In experimental FSGS induced with a cytotoxic podocyte antibody and in the BTBR ob/ob mouse model of DN, PEC-derived protein staining was significantly increased in PECs. Dual staining also showed de novo expression of the podocyte-specific ECM proteins laminin-β2 and agrin in PECs. Similar findings were observed in biopsies from patients with FSGS and DN. Increases in individual ECM proteins colocalized with CD44 in PECs in disease. To determine the role of CD44, FSGS was induced in CD44−/− and CD44+/+ mice. PEC staining for perlecan, collagen type IV-α2, laminin-β2, and agrin were significantly lower in diseased CD44−/− mice compared with diseased CD44+/+ mice. These results show that in experimental and human FSGS and DN, PECs typically in an activated state, produce both PEC-derived and podocyte-specific ECM protein isoforms, and that the majority of these changes were dependent on CD44.

193CHARACTERIZATION OF PORCINE TROPHECTODERM-DERIVED CELLS IN THE PRESENCE OF HBMP4 IN THE ABSENCE OF A FEEDER LAYER

2004 ◽  
Vol 16 (2) ◽  
pp. 218 ◽  
Author(s):  
K.B. Stewart ◽  
A.M. Adams ◽  
S.L. Pratt ◽  
S.L. Stice
Keyword(s):  
Extracellular Matrix ◽  
Cell Growth ◽  
Cell Biology ◽  
Collagen Type ◽  
Growth Characteristic ◽  
Calf Serum ◽  
Embryonic Stem ◽  
Collagen Type Iv ◽  
Type Iv ◽  
Embryonic Disc

A porcine trophoblastic cell line could provide a powerful model for understanding trophoblast cell biology as well as placental gene expression and proteomics in vitro. In this experiment, we derived porcine trophoblastic cells from trophectoderm tissue and assessed their growth on three different extracellular matrix substrates and in three different concentrations of human recombinant bone morphogenetic protein 4 (hBMP4). Human BMP4 has been shown to induce differentiation of human embryonic stem cells into trophoblast lineages. Elongated embryos were flushed using DPBS supplemented with 1% fetal calf serum and penicillin-streptomycin (1X) from the hysterectomized uteri of superovulated and bred prepuberal gilts 15 days post-insemination. The embryonic disc was visualized with a dissecting microscope. The trophectoderm tissue was cut 2–3mm away from the embryonic disc with a scalpel and the trophectoderm tissue was manually dissected into cell aggregates. These aggregates were plated on collagen type IV, Matrigel, and human extracellular matrix (laminin, collagen type IV and heparan sulfate proteoglycan derived from human placenta) in culture medium (DMEM with 15% FCS, 0.1mM 2-mercaptoethanol, 4ngmL−1 basic FGF4 and 1X P/S) in the presence or absence of hBMP4 at 0, 10, or 20ngmL−1. Cell outgrowth was observed within 24 hours of culture. After three days of culture, various cell types (based on size and morphology) were present. Among cultures of predominant large cells were colonies of smaller cells with epithelial-type morphology that had a prominent nucleus and a high nuclear-to-cytoplasmic ratio. The epithelial-type cells grew in tight colonies with definite borders and contained cytoplasmic structures resembling lipid-containing vesicles. These colonies initially appeared on all matrices across all hBMP4 concentrations. After seven days in culture the colonies developed distinct differences across groups. Cell growth on collagen was comprised of tight colonies having definite borders among large cells. Colonies on collagen were larger and more pronounced in both the hBMP4-supplemented groups than when cultured without hBMP4. The Matrigel coated plates contained large sheets of epithelial-type cell growth instead of compact colonies. This type of growth characteristic was present in all hBMP4 treatments on Matrigel. In contrast, few cells survived and propagated on human extracellular matrix. Only small colonies having the desired morphology were among the large cells on human extracellular matrix when cultured in medium containing 10ngmL−1 hBMP4. Cells were passaged and only cells growing on Matrigel could be further cultured. These data suggest that both the cell substrate and hBMP4 affect initial trophoblast outgrowths. Further analysis including immunocytochemistry and RT-PCR is currently being performed to better characterize these cells. Epidemiology/Diseases

scholarly journals Morphometric analysis of the human endoneurial extracellular matrix components during aging

2021 ◽  
Vol 73 (1) ◽  
pp. 103-110
Author(s):  
Braca Kundalic ◽  
Sladjana Ugrenovic ◽  
Ivan Jovanovic ◽  
Vladimir Petrovic ◽  
Aleksandar Petrovic ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Collagen Type ◽  
Linear Regression Analysis ◽  
Collagen Type I ◽  
Nerve Fibers ◽  
Collagen Type Iv ◽  
Type I ◽  
Type Iv ◽  
Ecm Proteins ◽  
Extracellular Matrix Components

The aim of this study was to analyze the expression of extracellular matrix (ECM) proteins in human endoneurium during aging. We harvested 15 cadaveric sural nerves, distributed in 3 age groups (I: 25-44, II: 45-64, III: 65-86 years old). Histological sections were stained immunohistochemically for the presence of collagen type I, type IV and laminin, and the ImageJ processing program was used in morphometrical analysis to determine the percentages of these endoneurial proteins. In two younger groups, the endoneurial matrix of the sural nerve was composed from about equal proportions of these proteins, which may be considered a favorable microenvironment for the regeneration of nerve fibers. Linear regression analysis showed a significant increase in endoneurial collagen type IV with age, while collagen type I and laminin significantly decreased during the aging process. In cases older than 65 years, remodeling of the endoneurial matrix was observed to be significantly higher for the presence of collagen type IV, and lower for the expression of collagen type I and laminin. This age-related imbalance of ECM proteins could represent a disadvantageous microenvironment for nerve fiber regeneration in older adults. Our findings contribute to the development of therapeutic approaches for peripheral nerve regeneration.

scholarly journals ADAMTS-family protease MIG-17 regulates synaptic allometry by modifying the extracellular matrix and modulating glia morphology during growth

2019 ◽  
Author(s):  
Tingting Ji ◽  
Kai Wang ◽  
Jiale Fan ◽  
Jichang Huang ◽  
Mengqing Wang ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Extracellular Matrix ◽  
Collagen Type ◽  
Collagen Type Iv ◽  
Genetic Screens ◽  
Genetic Studies ◽  
C Elegans ◽  
Type Iv ◽  
Forward Genetic Screens ◽  
Signaling Axis

ABSTRACTSynapses are largely established during embryogenesis and maintained during growth. The mechanisms that regulate synaptic allometry—the maintenance of synaptic positions during growth—are largely unknown. We performed forward genetic screens inC. elegansfor synaptic allometry mutants and identifiedmig-17, a secreted metalloprotease of the conserved ADAMTS family. Through proteomic mass spectrometry analyses, cell biological and genetic studies we determined that MIG-17 is expressed by muscle cells to modulate glia location and morphology. Glia are proximal to synapses, and the glial location and morphology determine synaptic position during growth.Mig-17regulates synapse allometry by influencing epidermal-glia crosstalk through the regulation of basement membrane proteins, including collagen type IV, SPARC and fibulin. Our findings underscore the importance of glia location in the maintenance of synaptic allometry, and uncover a muscle-epidermal-glia signaling axis, mediated through the extracellular matrix, in the regulation of glia morphology and synaptic positions during growth.

scholarly journals Recombinant human bone morphogenetic protein 2B stimulates PC12 cell differentiation: potentiation and binding to type IV collagen.

1992 ◽  
Vol 119 (6) ◽  
pp. 1721-1728 ◽  
Author(s):  
V M Paralkar ◽  
B S Weeks ◽  
Y M Yu ◽  
H K Kleinman ◽  
A H Reddi
Keyword(s):  
Extracellular Matrix ◽  
Cell Differentiation ◽  
Bone Morphogenetic Protein ◽  
Collagen Type ◽  
Activin A ◽  
Collagen Type Iv ◽  
Type Iv ◽  
Morphogenetic Protein ◽  
Extracellular Matrix Components ◽  
Matrix Components

Bone morphogenetic protein 2B (BMP 2B, also known as BMP 4) induces cartilage and bone morphogenesis in ectopic extraskeletal sites. BMP 2B is one of several bone morphogenetic proteins which along with activins and inhibins are members of the transforming growth factor-beta (TGF-beta) family. Both BMP 2B and activin A, but not TGF-beta 1, induce rat pheochromocytoma PC12 neuronal cell differentiation and expression of VGF, a nervous system-specific mRNA. PC12 cells exhibited approximately 2,500 receptors per cell for BMP 2B with an apparent dissociation constant of 19 pM. Extracellular matrix components, including fibronectin, laminin, and collagen type IV potentiated the activity of BMP and activin A, with the latter being the most active. Direct experiments demonstrated that radioiodinated BMP 2B bound to collagen type IV better than to either laminin or fibronectin. These data demonstrate a common neurotrophic activity of both BMP 2B and activin A, and suggest that these regulatory molecules alone and in conjunction with extracellular matrix components may play a role in both the development and repair of nervous tissue.

scholarly journals Migration through the Extracellular Matrix by the Parasitic Protozoan Leishmania Is Enhanced by Surface Metalloprotease gp63

2003 ◽  
Vol 71 (2) ◽  
pp. 1008-1010 ◽  
Author(s):  
Bradford S. McGwire ◽  
Kwang-Poo Chang ◽  
David M. Engman
Keyword(s):  
Extracellular Matrix ◽  
Collagen Type ◽  
Collagen Type Iv ◽  
Parasitic Protozoan ◽  
Type Iv ◽  
Extracellular Matrix Components ◽  
Matrix Components

ABSTRACT Leishmania species engineered to express high levels of the surface metalloprotease gp63 have enhanced capacity of migration through extracellular matrix in vitro. This correlates with gp63 degradation of extracellular matrix components, such as collagen type IV and fibronectin, and suggests an important role for gp63 in the pathogenesis of leishmaniasis.

Appearance of the Myofibroblastic Phenotype in Dupuytren’s Disease Is Associated with a Fibronectin, Laminin, Collagen Type IV and Tenascin Extracellular Matrix

Pathobiology ◽  
1994 ◽  
Vol 62 (2) ◽  
pp. 55-58 ◽  
Author(s):  
Alexander Berndt ◽  
Hariwig Kosmehl ◽  
Detlef Katenkamp ◽  
Volker Tauchmann
Keyword(s):  
Extracellular Matrix ◽  
Collagen Type ◽  
Collagen Type Iv ◽  
Type Iv

scholarly journals Enterococcus faecalis Adhesin, Ace, Mediates Attachment to Extracellular Matrix Proteins Collagen Type IV and Laminin as well as Collagen Type I

2000 ◽  
Vol 68 (9) ◽  
pp. 5218-5224 ◽  
Author(s):  
Sreedhar R. Nallapareddy ◽  
Xiang Qin ◽  
George M. Weinstock ◽  
Magnus Höök ◽  
Barbara E. Murray
Keyword(s):  
Extracellular Matrix ◽  
Enterococcus Faecalis ◽  
Collagen Type ◽  
Collagen Type I ◽  
Immune Serum ◽  
Collagen Type Iv ◽  
Type I ◽  
Collagen Types ◽  
Type Iv ◽  
Ecm Proteins

ABSTRACT Adhesin-mediated binding to extracellular matrix (ECM) proteins is thought to be a crucial step in the pathogenic process of many bacterial infections. We have previously reported conditional adherence of most Enterococcus faecalis isolates, after growth at 46°C, to ECM proteins collagen types I and IV and laminin; identified an E. faecalis-specific gene, ace, whose encoded protein has characteristics of a bacterial adhesin; and implicated Ace in binding to collagen type I. In this study, we constructed an ace disruption mutant from E. faecalis strain OG1RF that showed marked reduction in adherence to collagen types I and IV and laminin when compared to the parental OG1RF strain after growth at 46°C. Polyclonal immune serum raised against the OG1RF-derived recombinant Ace A domain reacted with a single ∼105-kDa band of mutanolysin extracts from OG1RF grown at 46°C, while no band was detected in extracts from OG1RF grown at 37°C, nor from the OG1RF ace mutant grown at 37 or 46°C. IgGs purified from the anti-Ace A immune serum inhibited adherence of 46°C-grown E. faecalis OG1RF to immobilized collagen type IV and laminin as well as collagen type I, at a concentration as low as 1 μg/ml, and also inhibited the 46°C-evoked adherence of two clinical isolates tested. We also showed in vitro interaction of collagen type IV with Ace from OG1RF mutanolysin extracts on a far-Western blot. Binding of recombinant Ace A to immobilized collagen types I and IV and laminin was demonstrated in an enzyme-linked immunosorbent assay and was shown to be concentration dependent. These results indicate that Ace A mediates the conditional binding of E. faecalis OG1RF to collagen type IV and laminin in addition to collagen type I.

scholarly journals Schwann cells use a novel collagen-dependent mechanism for fibronectin fibril assembly

1998 ◽  
Vol 111 (18) ◽  
pp. 2763-2777 ◽  
Author(s):  
M.A. Chernousov ◽  
R.C. Stahl ◽  
D.J. Carey
Keyword(s):  
Extracellular Matrix ◽  
Schwann Cells ◽  
Collagen Type ◽  
Collagen Type I ◽  
Collagen Type Iv ◽  
Type I ◽  
Type Iv ◽  
The Matrix ◽  
Fibronectin Fibrils ◽  
Dependent Mechanism

Cultured rat Schwann cells were stimulated to deposit fibrillar extracellular matrix by treatment with ascorbic acid in the absence of nerve cells. Immunofluoresence staining of the matrix showed that it contains collagens types I and IV, fibronectin and perlecan but not laminin. Collagen type IV, fibronectin and perlecan co-distributed completely in the matrix fibrils, whereas collagen type I was present in only a subset of these fibrils. Time course studies indicated that collagen type I fibrils appear at late stages of matrix formation. Digestion of Schwann cell extracellular matrix with collagenase effectively disrupted most of the matrix including fibronectin fibrils. This was in contrast with fibroblasts, where collagenase treatment removed collagen with no visible effect on fibronectin fibrils. alpha5 integrin was expressed on the cell surface of Schwann cells and partially codistributed with fibronectin-containing fibrils. This suggests that the inability of Schwann cells to deposit fibronectin-containing matrix through a conventional, collagen-independent mechanism was not due to the lack of fibronectin-binding integrins on their cell surface. Polyclonal anti-fibronectin antibodies inhibited the deposition of fibronectin into the matrix fibrils, whereas collagen type IV fibrils were generally unaffected. Growth of Schwann cells on collagen type IV-coated substrate in the absence of ascorbate induced deposition of fine fibronectin fibrils. These results suggest that Schwann cells use an apparently novel, collagen type IV-dependent mechanism for the deposition of fibronectin into their extracellular matrix.

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