Suppression of the release of type-1 plasminogen activator inhibitor from human vascular endothelial cells by Hawaii deep sea water  Shigeru Ueshima, Hideharu Fukao, Kiyotaka Okada, Osamu Matsuo  Pathophysiology  Volume 9, Issue 2, Pages 103-109 (January 2003) DOI: 10.1016/S0928-4680(02)00076-7

Fig. 1 Release of PAI-1 antigen into the CM of HUVECs cultured with control (closed column) or HDSW medium (hatched column) at semi-confluent or confluent stage. HUVECs were cultured with either control medium or HDSW medium. Then HUVECs at semi-confluent or confluent state were incubated with serum-depleted control medium or serum-depleted HDSW medium for 12 h. The amount of PAI-1 antigen in the CM was measured by PAI-1 ELISA. Values are expressed as mean±S.D. of three wells in duplicate experiments. **P<0.01. Pathophysiology 2003 9, 103-109DOI: (10.1016/S0928-4680(02)00076-7)

Fig. 2 Release of t-PA antigen into the CM of HUVECs cultured with control (closed column) or HDSW medium (hatched column). HUVECs were cultured with either control medium or HDSW medium. Then HUVECs at confluent state were incubated with serum-depleted control medium or serum-depleted HDSW medium for 12 h. The amount of t-PA antigen in the CM was measured by t-PA ELISA. Values are expressed as mean±S.D. of three wells in duplicate experiments. *P<0.05. Pathophysiology 2003 9, 103-109DOI: (10.1016/S0928-4680(02)00076-7)

Fig. 3 t-PA, u-PA and PA activities in the CM of HUVECs cultured with control (closed column) or HDSW medium (hatched column). HUVECs were cultured with either control medium or HDSW medium. Then HUVECs at confluent state were incubated with serum-depleted control medium or serum-depleted HDSW medium for 12 h. t-PA (A), u-PA (B) and PA (C) activities in the CMs were measured by using S-2288, S-2444 and S-2251, respectively. And each activity was revealed as optical density at 405 nm of wave length, which was generated by amidolysis of each chromogenic substrate. Values are expressed as mean±S.D. of three wells in duplicate experiments. *P<0.05. Pathophysiology 2003 9, 103-109DOI: (10.1016/S0928-4680(02)00076-7)

Fig. 4 Northern blot of PAI-1 mRNA. (A) Northern blot of mRNAs for PAI-1 mRNA and GAPDH mRNA. (B) Each PAI-1 signal was normalized by corresponding GAPDH signal, densitometrically. Values are expressed as mean±S.D. of three independent experiments. Lane 1: cultured with control medium for 3 days to form confluent and then exchanged with fresh control medium to incubate for further 3 days, lane 2: same as lane 1 except using HDSW medium, lane 3: cultured with control medium for 3 days to form confluent and then exchanged with fresh HDSW medium to incubate for further 3 days, lane 4: cultured with HDSW medium for 3 days to form confluent and then exchanged with fresh control medium to incubate for further 3 days. Pathophysiology 2003 9, 103-109DOI: (10.1016/S0928-4680(02)00076-7)

Fig. 5 Lysis of 125I-fibrin clot on HUVECs. Degradation of 125I-fibrin clot on HUVECs which had been cultured with control medium for 6 days (●-●), control medium for 3 days and then HDSW medium for 3 days (○-○), HDSW medium for 6 days (■-■), and HDSW medium for 3 days and then control medium for 3 days (□-□), after adding with plasminogen (12 μg/ml) was monitored as released radio-activities of 125I-fibrin degradation products. The spontaneous release of radioactivity in the presence of HUVECs is shown (▵-▵). Pathophysiology 2003 9, 103-109DOI: (10.1016/S0928-4680(02)00076-7)