Abstract: Objective Pro-urokinase,a new thrombolytic agent that is currently being evaluated for the treatment of myocardial infarction,stroke and pulmonary embolism.However little research has been done about the effects of recombinant pro-urokinase on endothelial cells.The purpose of this article is to study the effects of pro-urokinase on the expression and release of urokinase type plasminogen activator receptor(u-PAR) and plasminogen activator inhibitor1(PAI-1) and to study the effects of pro-urokinase on the mRNA expression of urokinase type plasminogen activator(u-PA) in human pulmonary arterial endothelial cells(HPAECs).Methods The HPAECs were cultured in M200 medium with low serum growth supplement until became confluent.Then the HPAECs were starvated for 12 h,and ready to be used in different experiments.(1) HPAECs were incubated with pro-urokinase(0 or 150 IU/mL) for 8 h,then the medium was centrifuged for 10 min at 1 000 r/min and expression of u-PAR or PAI-1 in the medium detected with ELISA kits.(2) HPAECs was incubated with pro-urokinase 150 IU/mL for 0,4,8,12 and 24 hours.In each group,the total RNA of HPAECs was extracted by Trizol reagent and the gene expression of u-PA mRNA detected with RT-PCR and compared with that of GAPDH mRNA.All cells in these experiments were of 3 to 6 passages.Results The cells were flat with round or oval nuclei containing several prominent nucleoli.Six to seven days after plating,the cultured HPAECs formed a monolayer.In the ELISA tests,the u-PAR content in pro-urokinase treated cell was statistically increased as compared with the control ones((0.51±0.04)μg/L vs(0.58±0.05)μg/L,P=0.005);the PAI-1 content in serum was statistically decreased in pro-urokinase treated cells as compared with control ones((66.75±7.92)μg/L vs(53.38±12.18)μg/L,P=0.009).In RT-PCR experiments,after incubation with pro-urokinase 150 IU/mL for increasing hours(0,4,8,12 and 24 hours),the u-PA bd/GAPDH band ratio was(0.34±0.11),(0.51±0.12),(0.58±0.12),(0.50±0.18) and(0.35±0.10) respectively.Pro-urokinase(150 IU/mL) incubation could up-regulate the expression of u-PA mRNA in HPAECs in a time-dependent manner and the highest expression was shown at 8 hours.Conclusion Under normal conditions,HPAECs cue could express or release u-PA,u-PAR,and PAI-1.Pro-urokinase could inhibit the release of PAI-1 and enhance the release of u-PAR from cell surface.Pro-urokinase could enhance the expression of u-PA mRNA in HPAECs in a time-dependent manner.Pro-urokinase has direct effects on the expression of u-PA system in HPAECs,which may thus enhance thrombolytic process.

Key words: recombinant pro-urokinase, pulmonary arterial endothelial cells, urokinase type plasminogen activator, urokinase type plasminogen activator receptor, plasminogen activator inhibitor-1, fibrinolysis