Continuous Vocalization during Kendo Exercises Suppresses Expiration of CO2.

One distinctive trait of kendo, the Japanese martial art of fencing, is the execution of sustained, high-effort vocalizations during actions. The purpose of this study was to determine the effect of these vocalizations on respiratory functions. First, the intensity of 3 kendo exercises was quantified by measuring oxygen uptake (V̇O2) and comparing it with V̇O2max measured during treadmill tests of 8 university kendo athletes. Respiratory variables of these 8 athletes were then analyzed using a portable breath gas analyzer during the most intensive kendo exercise, kakari-keiko, with and without vocalization. Breathing frequency (fB) increased regardless of vocalization, but in trials with vocalization, fB and ventilation were significantly lower, and expiration time was significantly longer. Components of expired gases were also affected by vocalization. Although there was no significant difference in oxygen uptake, vocalization yielded a reduction in carbon dioxide output (V̇CO2) and an increase in fraction of end-tidal carbon dioxide (FetCO2). We thus conclude that these vocalizations greatly affect expiration breathing patterns in kendo. Moreover, repetition of kakari-keiko caused a reduction in V̇CO2 and an increase in FetCO2 and CO2 storage. We consider the possibility that the sustained high-effort vocalizations of kendo also increase cerebral blood flow.

Microarray-based gene expression profiling of peripheral blood mononuclear cells in dairy cows with experimental hypocalcemia and milk fever.

Although a molecular diagnostic assay using clinically accessible tissue, such as blood, would facilitate evaluation of disease conditions in humans and animals, little information exists on microarray-based gene expression profiling of circulating leukocytes from clinically hypocalcemic cows. Therefore, peripheral blood mononuclear cells from dairy cows with experimentally induced hypocalcemia or spontaneous milk fever were subjected to oligo-microarray analysis to identify specific biomarker genes. In experimental hypocalcemia induced by a 4-h infusion of 10% disodium EDTA (n=4), 32 genes were significantly up- or downregulated compared with control treatment (4-h infusion of 11% calcium EDTA; n=4). In cows with milk fever (n=8), 98 genes were expressed differentially (either up- or downregulated) compared with healthy parturient cows (n=5). From these data, the following 5 genes were selected as being strongly related to both experimental hypocalcemia and milk fever: protein kinase (cAMP-dependent, catalytic) inhibitor ß (PKIB); DNA-damage-inducible transcript 4 (DDIT4); period homolog 1 (PER1); NUAK family, SNF1-like kinase, 1 (NUAK1); and expressed sequence tag (BI537947). Another gene (neuroendocrine secretory protein 55, NESP55) was also determined to be specific for milk fever, independently of hypocalcemia. The mRNA expression of these 6 genes in milk fever cases was verified by quantitative real-time reverse-transcription PCR and was significantly different compared with their expression in healthy parturient cows. In the present study, the selected genes appeared to be candidate biomarkers of milk fever because the continuous interactions between blood cells and the entire body suggest that subtle intracellular changes occur in association with disease. However, before any genomic biomarkers are incorporated into clinical evaluation of the disease, the effect of hypocalcemia on the mRNA expression of these genes in the tissues that regulate calcium homeostasis in dairy cows should be determined.

Analysis of circulating microRNA during early gestation in Japanese black cattle.

Circulating microRNAs (miRNAs) have been used as biomarkers for various diseases and physiological conditions in humans and mice; studies in domestic animals, particularly cattle, are limited. The importance of early pregnancy diagnosis (especially within the 21-d cow estrous cycle) in the livestock industry is extremely high. This study compared the circulating miRNAs in bred non-pregnant and pregnant Japanese Black cows, explored miRNAs as biomarkers for early pregnancy diagnosis, and established a measurement system that included selecting an appropriate reference miRNA and determining the effect of hemolysis on miRNA quantification in plasma. miRNA was extracted from the plasma of Japanese Black cows on day 21 after artificial insemination and subjected to a customized bovine oligonucleotide microarray for expression analysis. Differentially expressed miRNAs and reference miRNA candidates were selected and validated using reverse transcription-quantitative PCR (RT-qPCR). An appropriate endogenous reference miRNA for normalization was selected using NormFinder software. To evaluate the effect of hemolysis on miRNA quantification, hemolyzed samples were prepared using plasma from four cows in the estrous cycle and subjected to RT-qPCR. A total of 124 miRNAs were detected in bovine plasma by microarray analysis in bred non-pregnant and pregnant cows. The levels of five circulating miRNAs were significantly higher in pregnant cows than in bred non-pregnant cows, and 24 miRNAs were detected only in the pregnant group. NormFinder analysis and RT-qPCR validation showed that miR-2455 was an appropriate reference miRNA in the plasma of bred non-pregnant and pregnant Japanese Black cows, and miR-19b, miR-25, miR-29a, and miR-148a were significantly higher in the pregnant group. These four circulating miRNAs did not change during the estrous cycle and were less affected by hemolysis. In the current study, we found four miRNAs, miR-19b, miR-25, miR-29a, and miR-148a, which were present at high levels in the plasma of pregnant Japanese Black cows. Since these miRNAs are less affected by hemolysis, they may potentially be used as biomarkers for early pregnancy diagnosis in cattle.

Newly identified interferon tau-responsive Hes family BHLH transcription factor 4 and cytidine/uridine monophosphate kinase 2 genes in peripheral blood granulocytes during early pregnancy in cows.

In cattle, interferon-stimulated genes (ISGs) such as ISG15, MX1, MX2, and OAS1 are known as classic ISGs that are highly involved in the implantation process. Various molecules play a crucial role in the mechanisms underlying ISG effects. Although microarray analyses have highlighted the expression of various molecules during the implantation period, these molecules remain incompletely characterized. In the present study, various specifically expressed genes were selected and their characteristics were examined. The microarray data from peripheral blood leukocytes derived from artificially inseminated cows and granulocytes obtained from embryo-transferred cows, respectively, were used to identify new ISG candidates. Seven common genes, including ISG15 and OAS1, were confirmed, but only 4 of the 5 genes were amplified by reverse transcription quantitative polymerase chain reaction. In addition, 3 expressed sequence tags (ESTs) exhibited significantly greater expression in granulocytes from pregnant cows than that observed in bred nonpregnant cows, and the expression in granulocytes increased after interferon-tau stimulation. Sequence alignment revealed similar sequences within 2 ESTs on the Hairy and enhancer of split (Hes) family basic helix-loop-helix transcription factor 4 (HES4) gene. An additional EST was identified as cytidine/uridine monophosphate kinase 2 (CMPK2). In silico analysis facilitated the identification of transcription factor-binding sequences, including an interferon-stimulated response element and interferon regulatory factor-binding sites, within the promoter region of HES4 and CMPK2. These genes may function as new ISGs in the context of implantation and may participate in the coordination of the feto-maternal interface in cows.

Coupling between the photo-excited cyclic π system and the 4f electronic system in a lanthanide single molecule magnet.

A new type of electronic interaction which couples two angular momenta, i.e. the angular momentum of a localized 4f system (J) and an orbital angular momentum generated in a cyclic π conjugated system by irradiation with a circularly-polarized light, has been identified in a lanthanide single molecule magnet.

Evaluation of interferon-stimulated genes in peripheral blood granulocytes as sensitive responders to bovine early conceptus signals.

Early detection of gestation is important in the bovine industry. New methods have been developed to detect gene expression in leucocytes induced by interferon-tau (IFNT) as gestation biomarkers. However, it is debatable which blood cell is suitable for detecting gene expression. This study was aimed at confirming whether granulocytes respond to IFNT specifically. Granulocytes and mononuclear cells (MNCs) from cows, and several types of bovine cultured cells, were treated with recombinant (r) IFNT and gene expression was analysed by quantitative real-time reverse transcriptase (RT)-PCR and microarray analysis. Expression levels of IFN receptors (R1 and R2) were approximately 30- to 900-fold higher in granulocytes than in other cultured cells, and 1.5- to 2.5-fold higher in MNCs than in granulocytes. Microarray analysis following a 2h recombinant IFNT (rIFNT) treatment revealed expression changes for 900 genes in granulocytes. Genes with expression changes included known IFN-stimulated genes (ISGs; ISG15, OAS1, MX1, and MX2). Eighteen genes were selected following granulocyte microarray analysis and their expression changes were confirmed in early gestation, which revealed that nine genes had significantly higher expression levels in pregnant than in non-pregnant animals. In conclusion, granulocytes specifically responded to rIFNT treatment and the resulting gene expression changes correlated with those in vivo. Microarray analysis indicated that various genes showed expression changes in rIFNT-treated granulocytes, which may result in the identification of alternate candidate genes for the early detection of gestation. These results strongly indicate that gene expression in granulocytes is a suitable tool to determine pregnancy status.

Effect of reduced coenzyme Q10 (ubiquinol) supplementation on blood pressure and muscle damage during kendo training camp: a double-blind, randomized controlled study.

AIM: This randomized controlled study was designed to examine the effects of reduced coenzyme Q10 (ubiquinol; CoQ10) supplementation on blood pressure (BP) and exercise-induced muscle damage in kendo athletes during a 4-day kendo training camp. METHODS: In a double-blinded manner, 32 young kendo athletes were randomly assigned to supplement with either placebo or CoQ10 (600 mg) daily for 11 days from 1 week prior to camp to end of camp. BP was measured every morning after waking up during the training camp. Blood samples were taken at 3 time points; 1 week and 1 day prior and upon completion of training camp at 17:30. Statistical analysis was performed by repeated-measures analysis of variance followed by Bonferroni/Dunn post-hoc tests. RESULTS: Before the training camp started, there were no differences in diastolic BP between these groups. However, after kendo training started, diastolic BP in the CoQ10 group was significantly lower than that in the placebo group (P<0.05). Plasma creatine kinase (CK) and myoglobin (Mb) concentrations were significantly increased in both groups during the camp (P<0.05), whereas there were no significant differences in CK and Mb between CoQ10 and placebo groups (CK: P=0.82, Mb: P=0.69). CONCLUSION: Oral supplementation with reduced form of CoQ10 (ubiquinol; Kaneka QHTM) showed a significant hypotensive effect in young male kendo athletes during a 4-day kendo training camp, although it did not significantly ameliorate kendo exercise-induced muscle damage.

Thrombomodulin induction by all-trans retinoic acid is independent of HL-60 cells differentiation to neutrophilic cells.

The expression of thrombomodulin (TM), an antithrombotic factor, was investigated during neutrophilic differentiation of the HL-60 human myeloblastic cell line treated with all-trans retinoic acid (ATRA) or dimethyl sulfoxide (DMSO). Differentiation of the cells into neutrophilic cells progressed in a time- and dose-dependent fashion with ATRA or DMSO, as confirmed by the characteristic appearance of nitroblue tetrazolium (NBT) reduction and phagocytic activities, without induction of nonspecific esterase activity. TM antigen and cofactor activity for thrombin-dependent protein C activation were not detected in untreated HL-60 cells and the cells cultured with DMSO, but were expressed in a time-dependent manner in the cells cultured with ATRA. The level of TM expression in the HL-60 cells was not dose-dependent on ATRA concentrations, but maximum TM expression was obtained at 10(-7) M ATRA. TM expression levels decreased in cells cultured with greater than 10(-6) M ATRA, although the extent of cell differentiation into neutrophilic cells progressed at the higher ATRA concentrations. Since the TM antigen levels in the ATRA-treated cells also paralleled the TM mRNA levels, the data suggests that TM induction in the HL-60 cells cultured with ATRA reflected the levels of TM biosynthesis and was independent of HL-60 differentiation into neutrophilic cells. It was postulated that the appearance of TM with cofactor activity in neutrophilic cells differentiated from leukemic cells may contribute to prevention of vascular thrombosis in differentiation therapy of patients with acute promyelocytic leukemia by ATRA.

Cloning and localization of heparanase in bovine placenta.

Heparanase (HPA) degrades heparan sulfate proteoglycan in the extracellular matrix. To understand its role during implantation and placental development in bovine placentae, we cloned and characterized a full-length cDNA encoding bovine HPA and identified HPA localization in placentae. A full-length bovine HPA cDNA was cloned with a 1635 nucleotide open-reading-frame corresponding to a protein of 545 amino acids. The predicted amino acid sequence shares 80.0% and 76.5% identity with human and rat HPA, respectively. In placentomes of 60 and 210 days' gestation, in situ hybridization demonstrated HPA mRNA expression in binucleate cells. Binucleate cells may be a source of HPA throughout gestation in bovine placentae; they may assume specific role(s) in foetal and maternal dialogue. Western blot analysis of bovine placental extracts (day 60) was performed using anti-bovine HPA antibody prepared by immunization of rabbits with synthetic peptide conjugate corresponding to amino acid residues 474-489 of bovine HPA; it showed two immunoreactive proteins with approximate molecular weights of 55kDa and 65kDa. Further, immunofluoresence double staining of HPA and placental lactogen (PL) revealed that binucleate cells expressing HPA had immunoreactivity of PL. These results suggest that HPA is specifically expressed in bovine placental binucleate cells and that it may take migratory roles in placentogenesis for degrading the extracellular matrix.

Cyclic AMP increases thrombomodulin expression on membrane surface of cultured human umbilical vein endothelial cells.

Dibutyryl-cyclic AMP (Bt2cAMP; final concentration 1-5 mM) or beraprost sodium (synthetic prostacyclin, 100 nM) enhanced the expression of thrombomodulin (TM; an anticoagulant factor of endothelial cells) on the membrane surface of cultured human umbilical vein endothelial cells up to 1.4 times over the control within 9 hrs after the treatment, while the expression fell below the control level at 12 hrs and thereafter. 8-Bromo-cAMP (final concentration 1-5 mM) or 3-isobutyl-1-methylxanthine (IBMX; an inhibitor of phosphodiesterase; final concentration 10-1000 microM) enhanced the expression of TM on the cell surface at 12 hrs after the treatment. The enhancement of TM expression caused by Bt2cAMP was inhibited by incubation with phorbol 12-myristate 13-acetate. These results suggest that cAMP stimulates expression of TM in the endothelial cells.

Vascularized bone grafts to the upper extremities.

From 1979 to 1995 in our clinic, vascularized bone grafting was performed in 29 patients with large bone defects, established nonunion, congenital pseudoarthrosis, or avascular necrosis in the upper extremity. Four patients had traumatic bone defects, six had posttraumatic nonunions, two had congenital pseudoarthroses, five had amputations, nine had defects following tumor resection, and three had other lesions. Reconstructed sites were the humerus in 7 patients, the radius in 12, the ulna in 2, both radius and ulna in 1, and the metacarpal and phalangeal bones in 7. Donor bones were fibula in 19 cases, radius in 6, scapula in 2, and medial condyle of the femur in 2. Postoperative circulatory disturbances and venous thrombosis resulted in revision surgery in two patients. Thrombectomy and reanastomosis to other veins were performed, and these flaps took successfully. No patients required additional bone grafts. The mean period required to obtain radiographic bone union was 4 months (fibula, 4.5 months; scapula, 3.5 months; radius, 2.6 months; medial condyle of the femur, 4 months). Vascularized fibula graft is indicated in patients with large bone defects in the humerus, radius, and/or ulna. The scapula is easy to transfer to the proximal humerus on its pedicle. This donor is indicated in young women because operative scars can be hidden. The radius is usually harvested with skin, and its use is indicated in patients with bone loss in the hand including thumb amputations. Thin corticoperiosteal graft from the femur is indicated in patients with established nonunion of the humerus and radius without significant bony defects.

[Treatment of Diphyllobothrium latum and Taenia saginata infections with aminosidine (author's transl)].

Recently, it has been known that the aminosidine has marked anthelmintic efficacy against tapeworm. In this investigation, aminosidine was used for treating 14 cases with Ciphyllobothrium latum infection and 5 cases with Taenia saginata infection. Aminosidine was administered orally in a single dose of 50 mg/kg, followed by a purge after the treatment. Fourteen patients with D. latum infection and 5 patients with T. saginata infection expelled long strobila in all cases. Although only 7 scolices of 18 worms of D. latum were found in the stool and no scolex of T. saginata was found, follow-up examination for a long period showed no evidence of remaining infection with one exception of D. latum. Mild nausea, vomiting and abdominal pain were observed in only one of 19 cases given aminosidine. But in the other 18 cases, no side effects were encountered. It was concluded that aminosidine is safe, effective therapeutic agent for the treatment of cestodiasis in man.

Dynamic expression of SOLD1 in bovine uteroplacental tissues during gestation.

INTRODUCTION: Secreted protein of Ly-6 domain 1 (SOLD1), a novel member of the Ly-6 superfamily, is present in the extracellular matrix of the mesenchyme in placental cotyledonary villi and is possibly involved in placental construction. OBJECTIVES: We investigated bovine SOLD1 expression in uteroplacental tissues with temporo-spatial patterning throughout gestation. METHODS: Placentomal and endometrial tissues during gestation were analyzed for SOLD1 mRNA levels by using quantitative RT-PCR. Tissue sections of placentomes and intercaruncular endometrium were used for determining SOLD1 mRNA and protein levels respectively with in situ hybridization and immunohistochemistry. RESULTS: SOLD1 mRNA was more strongly expressed in fetal membranes than in endometrial tissues on day 35 of pregnancy, and its expression was maintained throughout pregnancy. SOLD1 mRNA was detected in mononucleate cells at early and mid gestation, and, interestingly, in mononucleate and binucleate trophoblast cells at late gestation. It was also present in endometrial epithelial cells and the stroma surrounding uterine glands. SOLD1 protein was widely distributed in the mesenchyme of the villous tree as the pregnancy progressed. CONCLUSION: Our study shows the temporo-spatial expression patterns of bovine SOLD1 during gestation in uteroplacental tissues. To our knowledge, this is the first report on the involvement of fetal trophoblastic and endometrial cells in the secretion of SOLD1. These results suggest that SOLD1 may play a crucial role not only in the remodeling of the uteroplacental area, but also in that of the endometrium during late gestation, in addition to its role at early gestation in placental construction.

Expression of ADAMTS1 mRNA in bovine endometrium and placenta during gestation.

A disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1) is a secreted protease. Through the regulation of extracellular matrix remodeling or developmental processes or both, ADAMTS1 is involved in several biological functions, including ovulation and embryo receptivity. However, the expression and possible role of ADAMTS1 in bovine endometrium is unknown. In this study, we analyzed ADAMTS1 mRNA expression in bovine endometrium during the estrous cycle, peri-implantation period, and at different stages of gestation by using quantitative real-time RT-PCR (qPCR) and in situ hybridization. The qPCR results indicated that the expression of ADAMTS1 mRNA was not affected by the day of the estrous cycle and was similar to cyclic levels on day 35 of gestation; however, the expression was more abundant in cotyledonary tissues of the placenta during late gestation. The in situ hybridization study showed that ADAMTS1 mRNA was detected mainly in uterine luminal epithelia and stromal cells during the estrous cycle and peri-implantation period. A disintegrin and metalloproteinase with thrombospondin motifs 1 mRNA was also expressed in the peri-implantation conceptus as well as in trophoblast cells, which include binucleate cells, and increased during late gestation. Furthermore, treatment of stromal cell with progesterone (300 nM) stimulated the expression of ADAMTS1 mRNA. This study indicates that ADAMTS1 participates in bovine endometrial remodeling, which is required for implantation and placental development in coordination with ovarian steroids.

Expression of extracellular matrix metalloproteinase inducer (EMMPRIN) and its expected roles in the bovine endometrium during gestation.

Extracellular matrix metalloproteinase inducer (EMMPRIN) and its induced matrix metalloproteinases (MMPs) play a crucial role in tissue remodeling during the peri-implantation period. However, the role of EMMPRIN in the bovine placenta is still unclear. We have postulated that EMMPRIN might play a regulatory role in trophoblastic cell functions during gestation by itself or through the regulation of MMP expression. In this study, EMMPRIN mRNA was detected in the bovine placentome and interplacentome throughout gestation, and its expression was significantly higher in the cotyledon during late gestation. In situ hybridization showed that EMMPRIN mRNA was expressed in the caruncular epithelium and the cotyledonary epithelium, including binucleate cells. Western blot analysis detected a band representing a protein of approximately 65 kDa in the caruncular and cotyledonary tissues, and the intensity of its expression was increased in both of these tissues during late gestation. The expression levels of MMP-2 and MMP-14 in the bovine placenta were higher during late gestation, as was observed for EMMPRIN. Therefore, EMMPRIN might regulate trophoblastic cell functions, especially those of binucleate cells, through MMP expression in the bovine placenta.

Expression of endogenous retrovirus-like transcripts in bovine trophoblastic cells.

UNLABELLED: Endogenous retrovirus envelope elements are considered to participate in trophoblastic cell fusion and multinucleate cell formation in humans, mice, and sheep. However, there is limited information about their roles in the ruminant placenta. OBJECTIVES: We explore and identify the endogenous retrovirus envelope element genes expressed in bovine trophoblasts. METHODS: The NCBI UniGene database (Build #97 Bos taurus) was screened by in silico analysis. After cloning endogenous retrovirus envelope element-like transcript (ERVE), expression profiles were analyzed with quantitative RT-PCR and in situ hybrizaidation. RESULTS: Two UniGene clusters, UniGene ID: Bt.68042 and Bt.85243, were detected, and ERVE-A gene was cloned. Weak expression of this gene was first detected on Day 20 of gestation, and the intensity of its expression increased up to Day 70 of gestation. The intensity of its expression was maintained throughout gestation in the placenta, and its specific expression in trophoblastic binucleate cells was confirmed by in situ hybridization. CONCLUSIONS: bERVE-A has a similar sequence to human syncytin-1, although it lacks an intact envelope sequence, and is specifically expressed in binucleate cells. This is the first evidence that endogenous retrovirus envelope element genes are expressed in bovine binucleate cells.

Oxidized low density lipoprotein reduces thrombomodulin transcription in cultured human endothelial cells through degradation of the lipoprotein in lysosomes.

Oxidized low density lipoprotein (LDL), a potent atherogenic lipoprotein, has been shown to cause the alteration of various endothelial functions. We have examined the effect of oxidized LDL on the cofactor activity for thrombin-dependent protein C activation and expression of thrombomodulin (TM), a cell surface antithrombotic glycoprotein, on cultured human umbilical vein endothelial cells. Oxidized LDL prepared by irradiation of LDL with 254-nm ultraviolet light did not directly affect the cofactor activity of isolated TM. Exposure of the cells to oxidized LDL (25-200 microg/ml), but not native LDL and acetylated LDL, reduced TM cofactor activity in parallel with its antigen levels on the cell surface in an oxidation-, concentration- and time-dependent manner. TM mRNA levels were reduced prior to decrease in TM antigen levels and were 50% of the control levels at 3.0 h after treatment of the cells with oxidized LDL. The apparent half-life time (t1/2 = 2.8 h) of TM mRNA in the oxidized LDL-treated cells, however, did not significantly differ from that (t1/2 = 2.6 h) in the control cells when the cells were coincubated with 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, a transcriptional inhibitor. Treatment of the cells with bafilomycin A1, an inhibitor for the proton pump of the lysosomes, inhibited intracellular degradation of the LDL and prevented down-regulations of the mRNA and the cell surface TM antigen levels caused by oxidized LDL. The inhibitor molecule in oxidized LDL was shown to be a lipid; organic solvent extracts (300 mg/ml cholesterol, an equivalent concentration with lipids in 200 microg/ml oxidized LDL) of oxidized LDL inhibited expression of TM antigen to nearly the same extent as the oxidized LDL, although water extracts did not affect TM expression on the cells. These results suggested that down-regulation of TM on endothelial cells exposed to oxidized LDL resulted from inhibition of its transcription mediated by lysosomal degradation of oxidized LDL and that a lipid component in the LDL could be an active species. A decrease in TM expression on the surface of endothelial cells may contribute to promote thrombosis in atherosclerotic lesions.

Flap transfers for the treatment of perichondrial ring injuries with soft tissue defects.

Seven cases (six fresh) of perichondrial ring injury with skin defects were treated using flap transfers. The study included four boys and three girls ranging in age from 2 to 9 years (average 6). They were followed up for an average of 8 years and 10 months. The period from injury to flap coverage was 8-12 days, with an average of 10 days in the fresh cases. Fracture was noted in four cases, with one an epiphyseal fracture. Peroneal flaps were transferred in four cases, latissimus dorsi myocutaneous flaps in two, and gastrocnemius muscle flap in one. Six flaps survived perfectly, and one failed due to venous thrombosis. This latter case was treated with a cross leg flap. Postoperative radiographic assessments confirmed partial growth plate arrest in the chronic case, but all the fresh cases had no postoperative growth disturbance. Flap coverage, for perichondrial ring injuries with wide skin defects, is a useful method not only for skin coverage, but for the prevention of growth disturbances as well.

Retinoic acid counteracts both the downregulation of thrombomodulin and the induction of tissue factor in cultured human endothelial cells exposed to tumor necrosis factor.

Inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) shift the hemostatic balance of endothelial cell surfaces in favor of prothrombotic properties by downregulating thrombomodulin (TM) and inducing tissue factor (TF) expression. We investigated the effects of retinoic acid (RA) on the prothrombotic properties of cultured umbilical vein endothelial cells exposed to TNF-alpha. The approximate 50% downregulation of TM antigen and cofactor activity induced by TNF-alpha (10 U/mL for 24 hours) was completely prevented when the cells were coincubated with both TNF-alpha and 10 mumol/L RA. In accordance with changes in cell surface TM antigen levels, the 70% decrease in TM messenger RNA (mRNA) induced by TNF-alpha was also prevented by 10 mumol/L RA. TNF-alpha induced TF activity of lysed cells (100-fold greater than untreated controls), an effect prevented when the cells were coincubated with both the TNF-alpha and 10 mumol/L RA. The 34-fold increase in TF mRNA levels induced by TNF-alpha (10 U/mL for 3 hours) was only two-fold in the presence of both TNF-alpha and RA. The effects of RA on the regulation of TM and TF expression in the cells exposed to TNF-alpha was dose-dependent from 0.01 to 10 mumol/L RA. The present results suggest that RA may affect on the mRNA level to alter TM and TF expression, effectively counteracting expression of prothrombotic properties of endothelial cells induced by inflammatory cytokines such as TNF-alpha.