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Endothelial cell growth factor methods of isolation and expression

a technology of growth factor and endothelial cells, applied in the field of new growth factor, can solve the problems of not being able to purify or characterize mitogenic activity, and no highly suitable mitogenic factor exists for this type of application, and achieve the effect of promoting re-endothelialization

Inactive Publication Date: 2006-02-02
FERRARA NAPOLEONE +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, no highly suitable mitogenic factor exists for this type of application.
To date, however, this mitogenic activity has not been purified or characterized.

Method used

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  • Endothelial cell growth factor methods of isolation and expression
  • Endothelial cell growth factor methods of isolation and expression
  • Endothelial cell growth factor methods of isolation and expression

Examples

Experimental program
Comparison scheme
Effect test

example a

[0235] Purpose of this Example was the determination of the molecular weight of the endothelial cell growth factor secreted in the medium by follicular cells.

[0236] Confluent cultures of follicular cells were incubated for three days in a serum-free medium consisting of low glucose Dulbecco's modified Eagle's medium supplemented with transferrin (10 μl / ml), insulin (5 μg / ml), 2 mM glutamine and antibiotics. The conditioned medium (CM) (150 ml) was then collected centrifuged (10000×g, 15 min. 4° C.) in order to remove cell debris, and then applied to a Heparin-Sepharose column which had been preequilibrated with 10 mM Tris / Cl, pH 7.0. The column was then sequentially eluted with 10 mM Tris / Cl, pH 7.0 containing 0.6, 1 and 3 M NaCl. The flow rate was 21 ml / h. Fractions of 700 μl were collected and aliquots were tested for bioactivity on adrenal cortex-derived microvascular endothelial cells. The majority of the bioactivity was eluted in the present of 0.6 M NaCl. This chromatographic...

example 1

Culture of Follicular Cells and Media Collection

[0240] Primary cultures of bovine pituitary FC were established as previously described (20,41). In one embodiment in the culturing the 20% fetal bovine serum in reference 20 was reduced to 10%. Concentrations of 5 to 20% should be effective. Also no DNAase is used. All other components are the same. At confluency, cells were passaged into large scale tissue culture plates in the presence of low glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM glutamine and antibiotics. Shortly after reaching confluency the cultures were extensively washed with PBS in order to remove serum components. The cells were then incubated in a serum-free medium consisting of DMEM plus transferrin (10 μl / ml), insulin (5 μg / ml), selenium (10−8 M), 2 mM glutamine and antibiotics. After three or four days, the medium was collected and replaced with fresh serum free medium. The collected medium was centrifuged (1000 ...

example 2

Concentration of Conditioned Medium

[0241] Four to six liter batches of conditioned medium (CM) were subjected to ammonium sulfate precipitation. Ammonium sulfate (500 g / L) was added under constant stirring, until the salt was completely in solution. After 8-12 hours in the cold room, the material was centrifuged (20,000×g, 45 min at 4° C.). The supernatant was discarded and the pellet was resuspended with 10 mM Tris / Cl, pH 7.2, 50 mM NaCl and dialyzed at 4° C. against the same buffer for 8-12 h. The final volume was 50-60 fold less than the original.

[0242] Alternatively, the CM is concentrated using ultrafiltration using an Amecon stir cell (2.5 liter unit) using a membrane having a molecular weight cut off of 10,000 daltons with similar results.

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Abstract

A novel growth factor specific for vascular endothelial cells has been identified in conditioned medium of bovine pituitary derived folliculo stellate cells. This factor, named folliculo stellate derived growth facto (FSdGF) or vascular endothelial growth factor (VEGF), was purified to homogeneity by a combination of heparin sepharose affinity chromatography, Bio Gel P-60 exclusion chromatography, Mono S ion exchange chromatography and hydrophobic chromatography on a C4 reverse phase HPLC column. The factor is also found in the murine AtT-20 cell line. Alternatively, the growth factor is purified by a first reverse phase HPLC using acetonitrile gradient followed by a second reverse phase HPLC using an isopropanol gradient. FSdGF, having a molecular weight of about 43,000 da, was characterized as a glycoprotein composed of two homologous sub units with MW of about 23 kDa. FSdGF was a potent mitogen for vascular endothelial cells with activity detectable at 10 pg / ml and saturation at 500 pg / ml. It did not stimulate the proliferation of other cell types such as bovine corneal endothelial cells, adrenal cortex cells, granulosa cells, BALB / MK cells or BHK-21 cells. Microsequencing revealed an amino terminal sequence containing no significant homology to any known protein. The release of FSdGF by pituitary cells and its unique target cell specificity indicate that FSdGF is useful in angiogenesis.

Description

BACKGROUND OF INVENTION [0001] The present application is a continuation-in-part of pending U.S. patent application Ser. No. 346,165, filed May 2, 1989, which is a continuation-in-part of pending U.S. patent application Ser. No. 328,181, filed Mar. 24, 1989, which are both incorporated by reference in its entirety. ORIGIN OF THE INVENTION [0002] The present invention was made with Government support in part on U.S. National Institute of Health Grant Nos. 5ROIEY 02186; 5ROIHL 20197; and HD 08035 awarded by the Department of Health and Human Services to the University of California at San Francisco, Calif. The U.S. Government has certain rights in this invention. [0003] 1. Field of the Invention [0004] The present invention relates to a novel growth factor for vascular endothelial cells identified in media condition of cultured bovine pituitary follicular cells and of murine tumor cells. The invention also relates to isolation and purification of the growth factor. [0005] 2. Descripti...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/18C07K14/59A61K38/00C07K14/52
CPCA61K38/1866C07K14/52
Inventor FERRARA, NAPOLEONEGOSPODAROWICZ, DENISPLOUET, JEAN
Owner FERRARA NAPOLEONE
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