Differential conversion of plasminogen to angiostatin by human corneal cell populations.
OBJECTIVE
Maintenance of avascularity of the normal cornea and control of neovascularization during wound healing depend on a balance of angiogenic and antiangiogenic factors. The purpose of this paper is to determine the ability of corneal cells to convert plasminogen to angiostatins and to compare these products with those made by intact corneas.


METHODS
RT-PCR was performed using plasminogen specific primers and the generated cDNA was sequenced. The proteins in corneal extracts, cornea conditioned medium, and medium from corneal epithelial cells, stromal fibroblasts, and myofibroblasts incubated with plasminogen were separated by SDS-PAGE and electroblotted. Western blots used monoclonal antibodies to kringles 1-3 to detect plasminogen and angiostatins. Angiostatins were isolated and tested for activity in a vascular endothelial cell proliferation inhibition assay.


RESULTS
Plasminogen, its mRNA and angiostatins were found in human corneal tissue extracts from the epithelial, stromal, and endothelial layers and from cornea conditioned medium, but not in medium from cultured epithelial cells, stromal fibroblasts, or myofibroblasts. However, cultures of corneal epithelial cells and stromal fibroblasts were able to convert exogenously added plasminogen to angiostatins, whereas cultured myofibroblasts did not. Angiostatins of 38 and 34 kDa were found under all angiostatin generating conditions; however other angiostatins differed in size. Further, the angiostatins isolated from fibroblast culture supernatants inhibited vascular endothelial cell proliferation.


CONCLUSIONS
Conversion of plasminogen to angiostatin is cell-type dependent. Because corneal cells generate angiostatins, use of human angiostatins may be a means of treating abnormal corneal neovascularization without the risk of side effects.
Warejcka
Debra J
DJ
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, USA. dwarejck@mcw.edu
Vaughan
Kimberly A
KA
Bernstein
Audrey M
AM
Twining
Sally S
SS
D043168:Angiostatins / Q000096:biosynthesis* / Q000235:genetics / Q000494:pharmacology; D015153:Blotting, Western; D018929:Cell Culture Techniques; D049109:Cell Proliferation / Q000187:drug effects; D003319:Corneal Stroma / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000166:cytology; D019573:Epithelium, Corneal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D009924:Organ Culture Techniques; D010958:Plasminogen / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D012333:RNA, Messenger; D043168:Angiostatins; D010958:Plasminogen