Endothelial cell growth factor methods of isolation and expression

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.

1 . Folliculo stellate-derived growth factor in isolated form.  
   
   
       2 . Folliculo stellate-derived growth factor of  claim 1  which is a dimeric protein having a molecular weight of approximately 43 kd, as determined by SS polyacrylamide electrophoresis under non-reducing conditions, and which comprises at its N-terminus the amino acid sequence  1 Ala-Pro-Met-Ala-Glu-Gly-Gly-Gln-Lys-Pro-His-Glu.  
   
   
       3 . Folliculo stellate-derived growth factor of  claim 2  which further comprises an internal amino acid sequence, obtainable upon tryptic digestion, selected from the group consisting of:  
     
       
         
               
               
             
                   
               
                 Ser-Phe-Cys-Arg-Pro-Ile-Glu-Thr-Leu-Val-Ssp-Ile- 
                   
               
                 Phe-Gln-Glu-Tyr-Pro-Asp-Glu-Ile; 
               
                 and 
               
                   
               
                 Ser-Phe-Cys-Arg-Pro-Ile-Glu-Thr-Leu-Val-Ssp-Ile- 
               
                 Phe-Gln-Glu-Tyr-Pro-Asp/Ile Glu. 
               
                   
               
           
              
              
              
              
              
              
              
              
             
          
         
       
     
   
   
       4 . A method of promoting the proliferation of endothelial cells which comprises applying to such cells a mitogenic amount of folliculo stellate-derived growth factor of  claim 1 .  
   
   
       5 . The method of  claim 4 , wherein the endothelial cells are grown in cell culture.  
   
   
       6 . A method of promoting vascular endothelialization which comprises applying to vascular surfaces of a host in need of such treatment an amount of folliculor stellate-derived growth factor of  claim 1  sufficient to promote endothelialization.  
   
   
       7 . The method of  claim 6 , wherein the folliculo stellate-derived growth factor is applied post-operatively to vascular surfaces following balloon angioplasty.  
   
   
       8 . The method of  claim 6 , wherein the folliculo stellate-derived growth factor is applied to the surfaces of vasculature and/or the surfaces of vascular grafts during or prior to vascular graft surgery.  
   
   
       9 . The method of  claim 6 , wherein the folliculo stellate-derived growth factor is administered to a host following myocardial infarction.  
   
   
       10 . A method of promoting wound healing which comprises administering to a wound an amount of the folliculo stellate-derived growth factor of  claim 1  sufficient to promote angiogenesis at the wound site.  
   
   
       11 . A pharmaceutical composition comprising the folliculo stellate-derived growth factor of  claim 1  and pharmaceutically acceptable carrier vehicle.  
   
   
       12 . A method for producing a substantially pure folliculo stellate-derived growth factor (FSdGF) in isolated form, which method comprises: 
 (a) providing a biological sample containing levels of FSdGF;    (b) extracting the solubilized portion of the sample;    (c) partially purifying the FSdGF from the extract using an aqueous salt-precipitation step;    (d) fractionating the purified extract using affinity chromatography employing heparin moieties linked to an insoluble support as the stationary phase and employing a salt gradient mobile phase of increasing salt concentration;    (e) fractionating the extract using gel exclusion chromatography;    (f) fractionating the sample using liquid chromatography; and optionally    (g) purifying the extract using reverse-phase high performance liquid chromatography.    
   
   
       13 . The method of  claim 12  wherein in step (c) the aqueous salt precipitation uses ammonium sulphate.  
   
   
       14 . The purified folliculo stellate-derived growth factor obtained by the method of  claim 12 .  
   
   
       15 . The folliculo stellate-derived growth factor in purified form which is a glycoprotein consisting essentially of two substantially homologous subunits each having a molecular weight of about 23,000 daltons.  
   
   
       16 . The folliculo stellate-derived growth factor of  claim 1  which is effective in wound healing in a human being at a concentration of between about 10 picogram/milliliter and about 500 picogram/milliliter.  
   
   
       17 . The method of  claim 12  wherein: 
 in the partially purifing step the FSdGF is contacted with aqueous ammonium sulfate solution;    in fractionating step (d) the heparin is attached to sepharose; and    in the purifying step (g) the reverse phase high performance liquid chromatography is conducted using an acetonitrile gradient.    
   
   
       18 . The purified growth factor produced by the method of  claim 17 .  
   
   
       19 . A folliculo stellate-derived growth factor in isolated form produced by a recombinant deoxyribonucleic acid (DNA) methods.  
   
   
       20 . The folliculo stellate-derived growth factor of  claim 19  having a molecular weight of about 43,000 da.  
   
   
       21 . Folliculo stellate-derived growth factor in isolated form.  
   
   
       22 . The folliculo stellate-derived growth factor of  claim 21  having a molecular weight of about 43 kDa.  
   
   
       23 . Folliculo stellate-derived growth factor of  claim 21  which is a protein having a molecular weight of approximately 43 to 45 kDa as determined by SS polyacrylamide electrophoresis under non-reducing conditions.  
   
   
       24 . Folliculo stellate derived growth factor of  claim 23  which comprises at its N-terminus the amino acid sequence  1 Ala-Pro-Met-Ala-Glu-Gly-Gly-Gln-Lys-Pro-His-Glu-Val-Val-Lys-Phe-Met-Asp-Val-Tyr-Gln.  
   
   
       25 . The folliculo stellate-derived growth factor of  claim 21  which under reducing conditions produces a substantially homologous dimer each unit having a molecular weight of about 23,000 daltons.  
   
   
       26 . A method of obtaining a concentrated folliculo stellate-derived growth factor in isolated form, which method comprises: 
 (a) obtaining a biological liquid supernatant sample containing levels of FSdGF using the conditioning medium of a culture;    (b) fractionating the sample of step (a) using heparin moities linked to an insoluble support as the stationary phase and using a salt gradient mobile phase of increasing salt concentration; and    (c) purifying the bioactive fraction of step (b) using gel electrophoresis.    
   
   
       27 . The method of  claim 26  wherein in the fractionating step the growth factor elutes at a sodium chloride concentration of between about 0.6 and 1.0 Molar.  
   
   
       28 . The method of  claim 27  wherein in the purifying step the gel is a suitable polyacrylamide.  
   
   
       29 . The folliculo stellate-derived growth factor produced by the method of  claim 26  having a molecular weight of about 43,000 da.  
   
   
       30 . A pharmaceutical composition comprising the folliculo stellate-derived growth factor produced by the method of  claim 26 .  
   
   
       31 . A pharmaceutical composition comprising the folliculo stellate-derived growth factor of  claim 21  and a pharmaceutically acceptable carrier vehicle.  
   
   
       32 . The pharmaceutical composition of  claim 21  wherein the carrier vehicle is a parenteral carrier vehicle.  
   
   
       33 . A method of obtaining a substantially pure folliculo stellate-derived growth factor (FSdGF) in isolated form, which method comprises: 
 (a) providing a biological sample containing levels of FSdGF;    (b) extracting the solubilized portion of the sample;    (c) partially purifying the FSdGF from the extract using an aqueous salt-precipitation step;    (d) fractionating the purified extract using affinity chromatography employing heparin moieties linked to an insoluble support as the stationary phase and employing a salt gradient mobile phase of increasing salt concentration;    (e) fractionating the extract using gel exclusion chromatography; and    (f) purifying the extract using reverse-phase high pressure liquid chromatography.    
   
   
       34 . The purified folliculo stellate-derived growth factor obtained by the method of  claim 33 .  
   
   
       35 . The purified folliculo stellate-derived growth factor of  claim 35  having a molecular weight of between about 43,000 and 45,000 da.  
   
   
       36 . A folliculo stellate-derived growth factor in purified form which is a glycoprotein consisting essentially of two substantially homologous subunits each having a molecular weight of about 23,000 daltons.  
   
   
       37 . The folliculo stellate-derived growth factor of  claim 21  which is effective in wound healing in a human being at a concentration of between about 10 picogram/milliliter and about 500 picogram/milliliter.  
   
   
       38 . The method of  claim 33  wherein in step (f) the reverse phase high pressure liquid chromatography is performed using an aqueous acetonitrile gradient.  
   
   
       39 . The method of  claim 33  which further includes: 
 (g) purifying the concentrated product of step (f) using reverse phase high performance liquid chromatography with an aqueous isopropanol gradient.    
   
   
       40 . The folliculo-stellate-derived growth factor of claims  20  having an N-terminus internal amino acid sequence of: Ala-Pro-Met-Ala-Glu-Gly-Gly-Gln-Lys-Pro-His-Glu-Val-Val-Lys-Phe-Met-Asp-Val-Tyr-Gln-(Arg)-Ser-Phe-X-Arg-Pro-Ile-Glu-Thr-Leu-(Val)-X-Ile-X -(Gln)-Glu-Tyr-(Pro)- wherein the amino acids in parenthesis are certain and the —X— indicates an amino acid of unknown identity.  
   
   
       41 . A process for producing human endothelial cell growth factor comprising, providing a replicable expression vector capable of expressing the DNA sequence encoding human endothelial cell growth factor in a suitable host, transforming said host to obtain a recombinant host, and maintaining said recombinant host under conditions permitting expression of said endothelial cell growth fator—encoding cDNA sequence to produce endothelial cell growth factor.  
   
   
       42 . The process according to  claim 41  including the further step of recovering said endothelial cell growth factor.  
   
   
       43 . The process according to  claim 42  wherein said expression vector is a bacteriophage.  
   
   
       44 . The process according to  claim 42  wherein said expression vector is plasmid.