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Tissue plasminogen activator is released into cultured medium by cultured human uveal melanocytes
Yafei Wang1, Dan-Ning Hu, Steven A McCormick
1Vision-Immunology Center, Department of Pathology, University of Connecticut Health Center, Farmington, CT, USA.
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.
Insights
Normal uveal melanocytes produce tissue plasminogen activator (t-PA), an enzyme crucial for cell invasion. This finding suggests uveal melanocytes contribute to intraocular processes like matrix remodeling and fluid dynamics.
Area of Science:
- Ocular oncology
- Cell biology
- Biochemistry
Background:
- Melanoma cells secrete tissue plasminogen activator (t-PA), a key factor in tumor invasion and metastasis.
- The production of t-PA by normal human uveal melanocytes has not been previously documented.
Purpose of the Study:
- To investigate t-PA production by cultured human uveal melanocytes.
- To compare t-PA production in uveal melanocytes with uveal melanoma and epidermal melanocytes.
Main Methods:
- Isolation and culture of human adult uveal melanocytes from donor eyes.
- Assay of plasminogen activator (PA) activity in conditioned media using amidolytic assays, fibrinography, and blocking antibodies.
- Western blot analysis to identify t-PA molecular weight and form.
Main Results:
- Free PA activity, predominantly t-PA, was detected in conditioned media from both normal uveal melanocytes and melanoma cells.
- Normal uveal melanocytes produced significantly more t-PA (3.23 IU/105 cells/24 h) than epidermal melanocytes (1.25 IU/105 cells/24 h), but less than uveal melanoma cells (11.0 IU/105 cells/24 h).
- Western blots confirmed the presence of one-chain t-PA (69 kDa) in conditioned media.
Conclusions:
- Uveal melanocytes contribute to the free t-PA activity observed in ocular fluids.
- This t-PA production by uveal melanocytes may influence intraocular matrix remodeling, fibrinolysis, and aqueous humor outflow.