title: Tissue plasminogen activator is released into cultured medium by cultured human uveal melanocytes.
abstract: Melanoma cells produce tissue plasminogen activator (t-PA) that plays an important role in tumor invasion and metastasis. The production of t-PA by normal human uveal melanocytes has not been reported previously. In order to explore this possibility, we studied the production of t-PA by cultured human uveal melanocytes and compared that with the production by cultured human uveal melanoma cells and epidermal melanocytes. Human adult uveal melanocytes were isolated and cultured from donor eyes. The cells were cultured in serum-free medium for 48 h and the conditioned medium then collected for the plasminogen activator (PA) activity assay. Free PA activity was tested in an amidolytic assay using a t-PA standard curve. PA type was identified by fibrinography and antihuman t-PA and urokinase plasminogen activator (u-PA) blocking antibodies. Free PA activity was found in the conditioned medium of normal melanocytes and melanoma cells. The predominant PA activity was t-PA. Normal uveal melanocytes produced more t-PA (3.23 +/- 0.73 IU/105 cells/24 h) than that of epidermal melanocytes (1.25 IU/105 cells/24 h) but much less than uveal melanoma cells (11.0 +/- 3.39 IU/105 cells/24 h). Western blot analysis revealed that most t-PA in conditioned media were one-chain t-PA with molecular weight of 69 kDa. Our study indicates that uveal melanocytes may contribute to the free t-PA activity previously found in aqueous humor and choroidal eye cup superfusions. Therefore, this function of uveal melanocytes may play a role in intraocular matrix remodeling, fibrinolysis and aqueous humor outflow.
authors.affiliation: Vision-Immunology Center, Department of Pathology, University of Connecticut Health Center, Farmington, CT, USA.
authors.forename: Yafei
authors.initials: Y
authors.lastname: Wang
authors.forename: Dan-Ning
authors.initials: DN
authors.lastname: Hu
authors.forename: Steven A
authors.initials: SA
authors.lastname: McCormick
authors.forename: Howard E
authors.initials: HE
authors.lastname: Savage
authors.forename: James
authors.initials: J
authors.lastname: O'Rourke
mesh_terms: D015153:Blotting, Western; D002478:Cells, Cultured / Q000166:cytology / Q000378:metabolism*; D017077:Culture Media, Conditioned / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism; D005341:Fibrinolysin / Q000378:metabolism; D006133:Growth Substances / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D008544:Melanocytes / Q000166:cytology / Q000378:metabolism*; D017433:Protein Structure, Secondary / Q000502:physiology; D010959:Tissue Plasminogen Activator / Q000378:metabolism*; D014602:Uvea / Q000166:cytology / Q000378:metabolism*
chemical_list: D017077:Culture Media, Conditioned; D006133:Growth Substances; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin