Regulatory Effect Of DDAH/ADMA System On Expression Of Tissue Factor In Endothelial Cells And Its Underlying Mechanisms

Bleeding is a prominent manifestation in the early phase of acuteleukemia and a major cause for mortality in patients with acute leukemia.Life-threatening hemorrhage is correlated with the elevated level of tissuefactor (TF) caused by leukemic cells. Besides constitutive expressinghigh TF antigen on the member of leukemia cells, inducible TFexpression on endothelial cells activated by interacting with leukemiccells may contribute to a significant increase in both TF mRNA level andprocoagulating activity in endothelial cells and pathogenesis of thecoagulopathy in patients with acute leukemia, disseminated intravascularcoagulation (DIC), which is characterized by the widespread activationof coagulation, occurs in patients with a variety of clinical conditionsincluding sepsis especially in patients with leukemia. Lipopolysaccharide(LPS), the most frequent causative bacteria component, can activateTF-mediated coagulation pathway and play a key role in pathogenesis ofDIC in patients with acute leukemia.Nitric oxide (NO), which is synthesized from L-arginine by NOsynthase (NOS) in normally functioning endothelial cells, exhibitsvarious biological properties including vasorelaxation, anti-inflammationand anti-thrombogenicity. Asymmetric dimethylarginine (ADMA), whichwas recently reported as a major endogenous NOS inhibitor, can inhibitNO production and induce endothelial dysfunction in vivo and in vitro.The plasma levels of ADMA were significantly elevated in severalcardiovascular diseases and were associated with patho-thrombogenesisin these states. But, there is no data about the plasma level of ADMA inpatients with acute leukemia and the relation between ADMA andcoagulopathy in leukemia. Previous studies showed endogenous NO onendotoxin or monocyte-induced TF expression in activated endothelialcells. Dimethylarginine dimethylaminohydrolase (DDAH) is the specifichydrolase of ADMA and plays a key role in modulation of ADMA levelboth in tissue and in cells. The DDAH/ADMA pathway has been recentlythought as a novel regulatory system of endogenous NO production andendothelia dysfunction. Therefore, one aim of the present study was to determine whether the DDAH/ADMA pathway is involved in LPS andleukemic cells-induced TF expression in endothelial cells.All-trans Retinoic Acid (ATRA) is the first choice drug to acutepromyelocytic leukemia (APL), which can relieve the,coagulopathy inpatients with AL quickly. Recently, ATRA was reported to upregulateDDAH mRNA expression and increase endogenous NO production inendothelial cells. Demethylbellidifolin (DMB) is a major xanthonecompound extracted from Swertia davidi Franeh (Gentianaceae), acommonly used Chinese medicinal herb, which has extensivepharmacological actions. Our previous work showed that DMB protectedagainst endothelial dysfunction induced by oxidative and inflammatorystimuli, and the protective effects of DMB on endothelium were related tothe reduction of ADMA concentration via improving DDAH activity. Aprevious study showed that a Xanthone compound prevented againstendotoxin-induced experimental DIC in rat. Based on these datamentioned above, our present study further determined whether ATRAand DMB can inhibit leukemic cells or LPS-induced TF expression inendothelial cells respectively, and these inhibition effects are related tothe modulation of the DDAH/ADMA pathway.The study includes two parts: (1) to determine the plasma levels ofADMA and TF antigen in patients with acute leukemia and regulation byDDAH/ADMA pathway of leukemic cells-induced TF expression inendothelial cells. (2) to study the role of the DDAH/ADMA pathway inLPS-induced TF expression in endothelial cells, furthermore, to study therole of the DDAH/ADMA pathway on the inhibitory effect of DMB onLPS-induced TF expression in endothelial cells.METHODS (1) One hundred and twenty-one cases of acuteleukemia and 20 healthy subjects were recruited; (2) Leukemic cells lineHL-60 and human umbilical vein endothelial cells (HUVECs) werecultured. The model of co-culturing was constructed with the two kindsof cell lines; (3) HUVECs were transfected with hDDAH cDNAexpressing plasmid to overexpress DDAH2; (4) Concentration of ADMAin plasma and in cultured media was measured by high-performanceliquid chromatography (HPLC). Activity of DDAH in endothelial cells was estimated by directly measuring the amount of ADMA metabolizedby the enzyme; (5) Plasma level of TF antigen was determined usingenzyme-linked immunosorbnent assay kits; (6) Cell viability was testedby MTT assay; (7) Total cell procoagulant activity was determined usinga one-stage clotting assay; (8) mRNA levels of TF and DDAH2 weredetected by reverse transcription PCR (RT-PCR); (9) Western blot wasused to determine protein expression of DDAH2; (10) Intracellular Ca²⁺concentration ([Ca²⁺]_i)were determined using fluoresent Ca²⁺ indicatorFura-2; (11) Intracellular reactive oxygen species (ROS) levels inendothelial cells were determined by H₂DCF-DA, an oxidant-sensitivefluorescent indicator; (12) DNA-binding activity of NF-κB in nuclearextracts from endothelial cells was analyzed by electrophoretic mobilityshift assay (EMSA).RESULTS (1) Plasma levels of ADMA in patients with newlydiagnosed acute leukemia were higher than those in healthy subjects aswell as antigen express of TF (P＜0.01). Plasma levels of ADMA inpatients with acute leukemia were significantly lower after treatment withchemotherapy (P＜0.01). Plasma levels of ADMA were significantlyhigher in patients with newly diagnosed acute leukemia as compared topatients of unremissioned and completely remissioned (P＜0.01). Nosignificant difference was found between cases of relapsed and de novoacute leukemia (P＞0.05). Statistical correlation was observed betweenthe plasma level of ADMA and antigen express of TF in 88 cases ofnewly diagnosed acute leukemia (R=0.853, P＜0.01). (2) Co-culture ofHL-60 with HUVECs could increase the TF-mediated procoagulatingactivity of mixed cells. Moreover, the increase in TF activity resultedfrom the induction of TF expression in endothelial cells, which wereevidenced by the fact that co-cultured media can markedly increase TFmRNA expression and activity in HUVECs but not in HL-60.Pre-incubation of HUVECs with 1μM ADMA for 6 h could significantlyenhance the increases in both TF mRNA level and activity induced byco-cultured media which were markedly attenuated in HUVEC withoverexpression of DDAH2. (3) [Ca²⁺]_i in endothelial cells were markedlyincreased after treatment with co-cultured media, and pretreatment with 1 μM ADMA could further enhance such increase in [Ca²⁺]_i whileoverexpression of DDAH2 could markedly attenuated the increase in[Ca²⁺]_i induced by co-cultured media. Intracellular Ca²⁺ chelatorBAPTA-AM could prevent the increases in both TF mRNA expressionand activity induced by co-cultured media in endothelial cell pretreatedwith ADMA. (4) Pre-incubation of HUVECS with ATRA (10^-7M～10^-5M) for 24h could concentration-dependently attenuate the increases inboth TF mRNA level and activity induced by co-cultured media.Moreover, the mRNA expression of DDAH2 was upregulated bytreatment with ATRA in a concentration-dependent manner. (5) LPS (0.01～1μg/ml) could induce TF expression in HUVECs in a concentration-and time-dependent manner. Exogenous ADMA (1μM) significantlyenhanced the increase in both TF mRNA level and activity induced byLPS (1μg/ml), whereas L-arginine (50 mM), overexpression of DDAH2and DMB markedly attenuated the LPS-induced TF increment. (6) Thelevel of ADMA in cultured mediurn was significantly elevated by LPSstimulation, concomitantly with a decrease in activity of DDAH. Sucheffects of LPS on DDAH/ADMA system were attenuated by DMB (1～10μM) in a concentration-dependent manner. (7) LPS could increaseintracellular ROS production and activate nuclear factor-κB (NF-κB),which were enhanced by exogenous ADMA (1μM) and attenuated byL-arginine (50 mM), overexpression of DDAH2 and DMB.CONCLUSIONS (1) Our present results found that plasma levelsof ADMA in patients with newly diagnosed acute leukemia wereremarkably elevated, and were positively correlated to antigen express ofTF in the same subjects. These data suggest that the elevated plasma levelof ADMA may contribute to the pathogenesis of the coagulopathy inpatients with acute leukemia. (2) The DDAH/ADMA pathway may beinvolved in leukemic cell-induced TF expression of endothelial cells viaregulating intracellular Ca²⁺ signaling. (3) ATRA could inhibit leukemiccell-induced TF expression of endothelial cells via modulatingendothelial DDAH/ADMA pathway. (4) The DDAH/ADMA pathway canregulate LPS-induced TF expression via ROS-NF-κB-dependent pathwayin endothelial cells. (5) DMB could inhibit LPS-induced TF expression at transcription level, and such effect of DMB might be related tomodulating DDAH/ADMA system and inhibiting ROS-NF-κB-dependentpathway.