scholarly journals Purification of a factor that promotes neurite outgrowth: isolation of laminin and associated molecules.

1985 ◽  
Vol 101 (3) ◽  
pp. 898-913 ◽  
Author(s):  
A D Lander ◽  
D K Fujii ◽  
L F Reichardt
Keyword(s):  
Endothelial Cells ◽  
Heparan Sulfate ◽  
Neurite Outgrowth ◽  
Conditioned Medium ◽  
Culture Medium ◽  
Cell Types ◽  
Cultured Neurons ◽  
Sulfate Proteoglycan ◽  
Corneal Endothelial Cells ◽  
Laminin Binding

When culture medium, conditioned by any of several cell types, is applied to a polycationic substratum, a substance is adsorbed that causes neurons cultured on that substratum to extend processes (neurites) rapidly and profusely. We have purified the factor responsible for this effect from medium conditioned by bovine corneal endothelial cells, and have shown that it is composed of the glycoprotein laminin and two associated laminin-binding molecules: a sulfated protein known as entactin, and a large heparan sulfate proteoglycan. Of these molecules, only laminin was found to be present throughout the purification in all fractions possessing neurite outgrowth-promoting activity and absent from all fractions lacking activity. Laminin, purified from other sources, has been shown previously to promote extensive outgrowth by cultured neurons. These and other data presented here support the conclusion that laminin is responsible for the neurite outgrowth-promoting activity of the conditioned medium factor. Evidence is also presented that the association of a proteoglycan with laminin promotes efficient attachment of laminin to polycationic substrata, particularly in the presence of competing molecules.

scholarly journals Characterization of a factor that promotes neurite outgrowth: evidence linking activity to a heparan sulfate proteoglycan.

1982 ◽  
Vol 94 (3) ◽  
pp. 574-585 ◽  
Author(s):  
A D Lander ◽  
D K Fujii ◽  
D Gospodarowicz ◽  
L F Reichardt
Keyword(s):  
Endothelial Cells ◽  
Neurite Outgrowth ◽  
Sympathetic Neurons ◽  
Cesium Chloride ◽  
Cell Types ◽  
Sulfate Proteoglycan ◽  
Corneal Endothelial Cells ◽  
Active Factor ◽  
Heparin Sulfate Proteoglycan ◽  
Heparin Sulfate

Rat sympathetic neurons, plated onto extracellular matrix produced by cultured bovine corneal endothelial cells, rapidly extended neurites in the absence of nerve growth factor (NGF). The response was unaffected by antiserum to NGF. Rapid outgrowth also occurred when sympathetic neurons were plated onto polylysine-coated surfaces that had been exposed to serum-free medium conditioned by corneal endothelial cells (CMSF). A response was seen even when the neurons were cultured without serum. When plated onto a polylysine-coated dish treated with CMSF over half its surface, only the neurons on the treated half extended neurites. The active factor in CMSF was destroyed by trypsin, acid (pH 1.6), base (pH 12.7), or heating to 80 degrees C; it was stable to heating to 60 degrees C, collagenase, deoxyribonuclease, and neuraminidase. The factor elutes just after the void volume of a Sepharose 6B column. In associative cesium chloride gradients, it sediments as a peak centered at a density of 1.36-1.37, corresponding to a peak of material that can be biosynthetically labeled with [35S]sulfate or [3H]leucine. Material from this fraction was inactivated by heparinase, but not chondroitinase ABC, implying that a heparin sulfate proteoglycan is essential for the factor's activity. Inactivation by contaminants in the heparinase preparation was ruled out. Further purification indicated that the active factor may exist as an aggregate containing a heparin sulfate proteoglycan and other molecules. CMSF also promoted neurite outgrowth by other types of neurons. Furthermore, a variety of cell types were shown to produce factors similar to that in CMSF.

The binding of heparin to the extracellular matrix of endothelial cells up-regulates the synthesis of an antithrombotic heparan sulfate proteoglycan

2008 ◽  
Vol 217 (2) ◽  
pp. 328-337 ◽  
Author(s):  
Edvaldo S. Trindade ◽  
Constance Oliver ◽  
Maria C. Jamur ◽  
Hugo A.O. Rocha ◽  
Célia R.C. Franco ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Heparan Sulfate ◽  
Heparan Sulfate Proteoglycan ◽  
Sulfate Proteoglycan

scholarly journals Characterization of the iPSC-derived conditioned medium that promotes the growth of bovine corneal endothelial cells

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e6734
Author(s):  
Qing Liu ◽  
Yonglong Guo ◽  
Shiwei Liu ◽  
Peiyuan Wang ◽  
Yunxia Xue ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Endothelial Cells ◽  
Conditioned Medium ◽  
Cell Morphology ◽  
Cell Cycle Progression ◽  
Cellular Proliferation ◽  
Activin A ◽  
Corneal Endothelial Cells ◽  
Induced Pluripotent

Corneal endothelial cells (CECs) maintain corneal transparency and visual acuity. However, the limited proliferative capability of these cells in vitro has prompted researchers to find efficient culturing techniques for them. The aim of our study was to evaluate the use of conditioned medium (CM) obtained from induced pluripotent stem cells (iPSCs) as a source for the effective proliferation of bovine CECs (B-CECs). In our study, the proliferative ability of B-CECs was moderately enhanced when the cells were grown in 25% iPSC conditioned medium (iPSC-CM). Additionally, hexagonal cell morphology was maintained until passage 4, as opposed to the irregular and enlarged shape observed in control corneal endothelial medium (CEM). B-CECs in both the 25% iPSC-CM and CEM groups expressed and Na+-K+-ATPase. The gene expression levels of NIFK, Na+-K+-ATPase, Col4A and Col8A and the percentage of cells entering S and G2 phases were higher in the iPSC-CM group. The number of apoptotic cells also decreased in the iPSC-CM group. In comparison to the control cultures, iPSC-CM facilitated cell migration, and these cells showed better barrier functions after several passages. The mechanism of cell proliferation mediated by iPSC-CM was also investigated, and phosphorylation of Akt was observed in B-CECs after exposure to iPSC-CM and showed sustained phosphorylation induced for up to 180 min in iPSC-CM. Our findings indicate that iPSC-CM may employ PI3-kinase signaling in regulating cell cycle progression, which can lead to enhanced cellular proliferation. Effective component analysis of the CM showed that in the iPSC-CM group, the expression of activin-A was significantly increased. If activin-A is added as a supplement, it could help to maintain the morphology of the cells, similar to that of CM. Hence, we conclude that activin-A is one of the effective components of CM in promoting cell proliferation and maintaining cell morphology.

Antiangiogenic antithrombin down-regulates the expression of the proangiogenic heparan sulfate proteoglycan, perlecan, in endothelial cells

Blood ◽  
2004 ◽  
Vol 103 (4) ◽  
pp. 1185-1191 ◽  
Author(s):  
Weiqing Zhang ◽  
Yung-Jen Chuang ◽  
Richard Swanson ◽  
Juan Li ◽  
Kyunga Seo ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Heparan Sulfate ◽  
Transforming Growth Factor ◽  
Limited Proteolysis ◽  
Heparan Sulfate Proteoglycan ◽  
Endothelial Cell Proliferation ◽  
Cycle Phase ◽  
Sulfate Proteoglycan ◽  
Down Regulation

Abstract Antithrombin, a key serpin family regulator of blood coagulation proteases, is transformed into a potent antiangiogenic factor by limited proteolysis or mild heating. Here, we show by cDNA microarray, semiquantitative reverse transcriptase–polymerase chain reaction (RT-PCR), Northern blotting, and immunoblotting analyses that the expression of the proangiogenic heparan sulfate proteoglycan (HSPG), perlecan, but not other HSPGs, is dramatically down-regulated in human umbilical vein endothelial cells (HUVECs) treated with antiangiogenic cleaved and latent forms of antithrombin but not with the native form. Down-regulation of perlecan expression by cleaved and latent antithrombins was observed in both basic fibroblast growth factor (bFGF)–stimulated and unstimulated cells, whereas the antiangiogenic antithrombins inhibited the proliferation of only bFGF-stimulated HUVECs by arresting cells at the G1 cell cycle phase. The importance of perlecan expression levels in mediating the antiproliferative effect of the antiangiogenic antithrombins was suggested by the finding that transforming growth factor-β1, a potent stimulator of perlecan expression in endothelial cells, blocked the down-regulation of perlecan expression and antiproliferative activity of cleaved antithrombin on endothelial cells. The previously established key role of perlecan in mediating bFGF stimulation of endothelial cell proliferation and angiogenesis suggests that a primary mechanism by which antiangiogenic antithrombins exert their effects is through the down-regulation of perlecan expression.

scholarly journals Heparan sulfate proteoglycan on leukemic cells is primarily involved in integrin triggering and its mediated adhesion to endothelial cells.

1996 ◽  
Vol 184 (5) ◽  
pp. 1987-1997 ◽  
Author(s):  
Y Tanaka ◽  
K Kimata ◽  
A Wake ◽  
S Mine ◽  
I Morimoto ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
T Cell ◽  
Cell Surface ◽  
Heparan Sulfate ◽  
Heparan Sulfate Proteoglycan ◽  
Leukemic Cells ◽  
Intercellular Adhesion Molecule ◽  
Proteoglycan Synthesis ◽  
Sulfate Proteoglycan ◽  
Heparin Binding

Leukocyte migration from circulation into tissue depends on leukocyte integrin-mediated adhesion to endothelium, but integrins cannot function until activated. However, it remains to be understood how tumor cells adhere to endothelium and infiltrate into underlying tissue. We studied mechanisms of extravasation of leukemic cells using adult T cell leukemia (ATL) cells and report the following novel features of cell surface heparan sulfate proteoglycan on ATL cells in ATL cell adhesion to endothelium: ATL cells adhere to endothelial cells through already activated integrins without exogenous stimulation; different from any other hematopoietic cells, ATL cells express a characteristic heparan sulfate capable of immobilizing heparin-binding chemokine macrophage inflammatory protein (MIP)-1 beta, a potent T cell integrin trigger, produced by the cells themselves; competitive interruption of endogenous heparan sulfate proteoglycan synthesis reduces cell surface MIP-1 beta and prevents ATL cells from integrin-mediated adhesion to endothelial cells or intercellular adhesion molecule-1 triggered through G-protein. We propose that leukemic cells adhere to endothelial cells through the adhesion cascade, similar to normal leukocyte, and that the cell surface heparan sulfate, particularly on ATL cells, is pivotally involved in chemokine-dependent autocrine stimulation of integrin triggering by immobilizing the chemokine on them.

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