Endothelin production by hypoxic human endothelium.

PURPOSE: The physiologic significance of endothelin remains incompletely defined. Procoagulant and antifibrinolytic activities are increased in hypoxic cultured human umbilical venous endothelial cells (HUVEC). We examined the effect of hypoxia on HUVEC endothelin-1 production in vitro to determine whether a correlation existed between the procoagulant and antifibrinolytic response to hypoxia previously observed and an increase in vasoconstrictor peptide secretion by hypoxic HUVEC. METHODS: Cultured HUVEC were rendered hypoxic (PO2 = 40 mm Hg) or control (PO2 = 120 mm Hg) for 24 hours. Media were either standard, 5 gm glucose/L (high glucose), or contained 500 units superoxide dismutase/ml (SOD). Endothelin-like immunoreactivity for endothelin-1 (ET-IR) in conditioned media was measured by radioimmunoassay and expressed as mean femtomoles per milliliter (+/- SD) per 100,000 cells. Viability of HUVEC was assessed by trypan blue exclusion. Significance was determined by use of Student's t test. RESULTS: Conditioned media from hypoxic cells contained 76% more ET-IR than was found in control counterparts (p < 0.004). The addition of high glucose or SOD did not diminish ET-IR; a trend to higher ET-IR was present in both these groups versus standard media (303% and 226%, respectively, p < 0.03). CONCLUSIONS: Thus 24 hours of hypoxia caused an increase in conditioned-media ET-IR in cultured HUVEC. Because SOD or greater substrate availability did not diminish endothelin presence in conditioned media, it seems that hypoxic induction of endothelin-1 production or secretion is signaled in a fashion unrelated to cell toxicity from the hypoxic period.

Hypoxia induces procoagulant activity in cultured human venous endothelium.

Although it normally presents a nonthrombogenic surface, endothelium is capable of procoagulant activity and suppression of native anticoagulant properties. We theorized that hypoxia could shift normal endothelium into a procoagulant state and tested this hypothesis in cultured human umbilical venous endothelial cells. Human umbilical venous endothelial cells were obtained from fresh umbilical cords. Passage two cells were placed in control (PO2 greater than 120 mm Hg) or hypoxic (PO2 less than 60 mm Hg) media and incubated in control or hypoxic environments for 24 hours. In additional experiments, cells were reoxygenated for 4 or 48 hours after the initial hypoxic period. Cells were then assayed for procoagulant activity expressed as thromboplastin unit equivalents per 100,000 cells based on a thromboplastin standard curve. Results are expressed as percent increase in thromboplastin unit equivalents/100,000 cells +/- standard error versus control. Statistical significance was assessed by paired t test with p less than 0.05 considered significant. More than 95% of cells in all experimental and control preparations were viable after completion of the protocols. No morphologic variation was noted among the control and hypoxic groups. For cells rendered hypoxic without reoxygenation, the mean increase in procoagulant activity for the group (n = 4) versus control was 77% +/- 13% (p = 0.01). In the hypoxia and 4-hour reoxygenation group (n = 4), the mean increase in procoagulant activity was 141% +/- 43% (p less than 0.05). In cells reoxygenated for 48 hours after hypoxia (n = 8), the mean increase in procoagulant activity was 198% +/- 34% (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin inhibition of endothelial prostacyclin production.

Graft thrombosis is a common cause of early graft failure in pancreatic transplantation, even in the absence of rejection. Altered endothelial cell (EC) production of thromboactive substances may play a primary role in this and other settings of thrombosis where intraluminal insulin concentrations are high. We therefore investigated the effect of insulin on EC production of prostacyclin (PGI2), a potent endogenous antagonist of platelet aggregation and vasodilator. Confluent monolayers of human umbilical vein endothelial cells (HUVEC) were incubated for 15 min in Hanks'/Hepes buffer containing 0, 50, 250, or 500 microU/ml of human insulin or 0, 500, 1000, or 2000 microU/ml of porcine insulin. PGI2 production was assessed by exposing the monolayers to either 20 microM arachidonic acid (stimulated) or arachidonic acid vehicle (basal) for an additional 15 min. Test buffers were then assayed by RIA for 6-keto-PGF1 alpha, the stable metabolite of PGI2. The results of these experiments indicate that human insulin inhibits both basal and arachidonic acid-stimulated production of PGI2 from HUVEC in a dose-dependent manner. Inhibition of stimulated production was significant at concentrations of 250-500 microU/ml. Porcine insulin also inhibited arachidonic acid-stimulated production of PGI2 from HUVEC in a dose-dependent manner. However, HUVEC were less sensitive to porcine insulin than to human insulin and a concentration of 2000 microU/ml was required for significant inhibition. We therefore conclude that insulin, in locally high concentrations, inhibits endothelial PGI2 production in vitro. The ability of insulin to alter the production of thromboresistant substances from endothelium may facilitate thrombosis in circumstances where counterregulatory mechanisms are disturbed by injury or transplantation.

Cyclosporine inhibits endothelial cell prostacyclin production.

Cyclosporine (CsA)-induced alterations in endothelial production of prostacyclin (PGI2) may be a contributing factor to the decreased renal blood flow and glomerular thromboses associated with CsA nephrotoxicity. This study was performed to determine the effects of clinically relevant doses of CsA on arachidonic acid-stimulated production of PGI2 by cultured human vein endothelial cells (HUVEC). Confluent monolayers of HUVEC were incubated at 37 degrees C under 5% CO2/95% air for 15 min in Hanks'/Hepes buffer containing 0.01-5.0 microM CsA in 0.01% absolute alcohol. PGI2 production was then stimulated by the addition of 20 microM arachidonic acid and further incubation for 15 min. Supernatants were assayed for 6-keto-PGF1 alpha by radioimmunoassay. The results of this experiment indicate that CsA had a dose-dependent effect on arachidonic acid-stimulated PGI2 production by HUVEC which varied linearly with the log of the dose (r = -0.48, P = 0.003). Concentrations of 0.01, 0.05, and 0.1 microM were stimulatory and 0.5, 1.0, and 5.0 microM were inhibitory. Separate experiments demonstrated that 0.1-5.0 microM CsA had no effect on cell viability (trypan blue exclusion) or growth. These results indicate that CsA inhibits arachidonic acid-stimulated production of PGI2 by HUVEC in a dose-dependent fashion beginning at concentrations comparable to the high end of the therapeutic range. This suggests that CsA-induced decreases in endothelial production of PGI2 may contribute to the development of CsA nephrotoxicity.