Differences in the synthesis of secreted proteins in human retinal endothelial cells of diabetic and nondiabetic origin.
Protein synthesis and deposition by vascular endothelial cells play an important role in the neovascularization seen in diabetic retinopathy. In the present study, we have compared the pattern of protein accumulation in human retinal endothelial cells derived from diabetic and nondiabetic individuals. Confluent cultures of retinal endothelial cells were incubated for 18 h with a mixture of radiolabeled methionine and cysteine. Under basal conditions, without the addition of growth factors, diabetic retinal endothelial cells accumulated less radiolabeled protein than did cells of nondiabetic origin. Both epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) enhanced protein accumulation in cells of diabetic origin, but not in cells of nondiabetic origin. Analysis of radiolabeled proteins in the secreted fraction by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) revealed prominent protein bands at 220 and 49.5 kD in both diabetic and nondiabetic cultures that were identified by immunoblot analysis as fibronectin and a mixture of secreted protein acidic and rich in cysteine (SPARC) and plasminogen activator inhibitor-1 (PAI-1), respectively. The levels of PAI-1 were higher in the secreted fractions of diabetic cultures than in nondiabetic cultures. SDS-PAGE and autoradiography of the secreted fraction also revealed two protein components of approximate molecular weight 440 and 78 kD, which were present in fractions of diabetic origin but absent in those of nondiabetic origin. Our studies support unique differences in protein expression in cells of diabetic vs. nondiabetic origin in response to EGF and bFGF and identify two proteins exclusively expressed by cells of diabetic origin.
Munjal
I D
ID
Department of Biochemistry, Meharry Medical College, Nashville, TN.
McLean
N V
NV
Grant
M B
MB
Blake
D A
DA
D000328:Adult; D002478:Cells, Cultured; D003545:Cysteine / Q000378:metabolism; D003930:Diabetic Retinopathy / Q000188:drug therapy / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000473:pathology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005136:Eye Proteins / Q000096:biosynthesis*; D005260:Female; D005346:Fibroblast Growth Factors / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D008297:Male; D008715:Methionine / Q000378:metabolism; D008875:Middle Aged; D008970:Molecular Weight; D012160:Retina / Q000378:metabolism* / Q000473:pathology
D005136:Eye Proteins; D005346:Fibroblast Growth Factors; D004815:Epidermal Growth Factor; D008715:Methionine; D003545:Cysteine