Biomedical subjects

K Kizaki

Publications and source records attributed to K Kizaki.

At least 19 recordsLinked to original sources

PubMed2004

Temporospatial expression of placental lactogen and prolactin-related protein-1 genes in the bovine placenta and uterus during pregnancy.

The anatomical location of binucleate cells (BNC) influences protein expression but not steroid synthesis in ruminants. In order to determine if BNC in disparate locations differentially express bovine placental lactogen (bPL) and prolactin-related protein-1 (bPRP-1), we quantitated bPL and bPRP-1 transcripts in placentomal (cotyledonary, caruncular) and interplacentomal (intercotyledonary, intercaruncular) tissues throughout pregnancy in the bovine using real-time reverse transcription PCR (RT-PCR) and in situ hybridization. Levels of both bPL and bPRP-1 transcripts at peri-implantation were significantly higher (P < 0.01) in the fetal membrane than in caruncular and intercaruncular tissues. Thereafter, mRNA for these related proteins demonstrated different spatial as well as temporal patterns of expression. Levels of bPRP-1 transcripts peaked at day 60 of pregnancy. Between day 60 and 100, bPRP-1 transcripts fell by approximately sevenfold (P < 0.01) in cotyledonary and intercotyledonary tissues, and fourfold in caruncular (P < 0.01) tissue. Levels of bPRP-1 transcripts remained low in the cotyledonary, intercotyledonary, and caruncular tissues until peripartum. In contrast, bPL expression in placentomes increased with progression of gestation (P < 0.01), but decreased in interplacentomal tissue around peripartum. To conclude, disparate patterns of bPRP-1 and bPL genes are transcribed in the placentomal and interplacentomal tissues during gestation in the bovine, suggesting that these prolactin-like hormones serve distinct functions and are regulated differently in the uteroplacental unit in this species.

Animals↗

PubMed2003

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.

Amino Acid Sequence↗

PubMed2002

Expression of prolactin-related protein I at the fetomaternal interface during the implantation period in cows.

The bovine placenta secretes multiple molecules during implantation and placentation, many of which are produced by binucleate cells. In this study, production of prolactin-related protein I (PRP-I), a member of the non-classical prolactin-related family, was investigated during the implantation period in cows. Expression of bovine PRP-I (bPRP-I) in the placentome was examined during the preimplantation (days 17-19), implantation (days 20-25) and post-implantation (days 30-60) periods by immunohistochemistry, immunofluorescence and in situ hybridization. During the preimplantation period, both bPRP-I and bovine placental lactogen (bPL) were undetectable in trophoblastic cells. Both bPRP-I mRNA and protein appeared first at day 20 of gestation in trophoblastic binucleate cells and multinuclear cells that might migrate into the endometrium and fuse to epithelium; however, no bPL was detected in binucleate cells at this time. After implantation, on day 30, both bPRP-I and bPL were detected in binucleate cells and were co-expressed in the same cells. These data indicate that bPRP-I may play a role before implantation and that bPRP-I may be an excellent marker for trophoblastic cell differentiation, as well as a candidate for pregnancy diagnosis.

Animals↗

PubMed2001

Neurohistochemical analysis of regeneration in rat peripheral nerve after end-to-side neurorrhaphy.

We investigated the regenerative capacity of motor nerves repaired by end-to-side or end-to-end neurorrhaphy, using choline-acetyltransferase (ChAT) activity measurement or histological analysis. The right medial gastrocnemius nerves (MGNs) of 62 male Fisher strain rats were transected and divided into three groups. In group 1, the distal ends of the MGN were coapted to the side of the lateral gastrocnemius nerve, using a Y-shaped silicone tube in end-to-side neurorrhaphy. In group 2, the nerve ends were reconnected by the traditional end-to-end technique. In group 3, the nerve ends were separated and remained unrepaired. The MGNs were sampled 1, 2, and 3 months postoperatively for histological examinations and ChAT activity measurement. The medial gastrocnemius muscle (MGM) was also sampled for histological evaluations. Axonal regeneration of MGN and the recovery of MGM to nearly normal histology and weight were observed in groups 1 and 2 3 months postoperatively. Although there were no significant differences in ChAT values between groups 1 and 2, the values were significantly larger than that of group 3 3 months postoperatively. These findings suggested that end-to-side neurorrhaphy would be an alternative treatment for peripheral nerve injury in certain clinical situations.

Animals↗

PubMed2001

Expression of heparanase mRNA in bovine placenta during gestation.

This study reports the identification and sequence of a partial cDNA for bovine heparanase and the expression of its mRNA in the placenta during gestation. The 364 amino acid residues deduced from the 1092 bp cDNA fragment share 81.9% and 80.5% identity with amino acid sequences of human and rat heparanase, respectively. Northern blot hybridization showed that two mRNAs (2.0 and 3.5 kb) are strongly expressed in placenta, and weakly expressed in the kidney, lung, spleen and non-pregnant uterus. In the placenta, these transcripts were detected in the cotyledon at all stages of gestation examined, and in the intercotyledonary fetal membrane and caruncle on day 60, day 120 and day 260. Quantitative real-time RT-PCR analysis showed very low expression of heparanase mRNA in the conceptus before implantation (day 17), but high expression in the cotyledon-containing fetal membrane (days 27-34) after implantation. Furthermore, heparanase mRNA was detected in the cotyledon, intercotyledonary fetal membrane and caruncle after days 60-64 of gestation. However, no significant expression of heparanase mRNA was observed in intercaruncular endometrium at all stages of gestation examined. These results demonstrate that heparanase mRNA is expressed in the placentome, indicating that heparanase may play a role in implantation, and in placental development and function.

Amino Acid Sequence↗

PubMed2000

Acceleration of thrombomodulin gene transcription by retinoic acid: retinoic acid receptors and Sp1 regulate the promoter activity through interactions with two different sequences in the 5'-flanking region of human gene.

The interactions between retinoic acid- (RA)-dependent transcriptional regulatory sequences of the 5'-untranslated region of the thrombomodulin gene and nuclear RA-responsive proteins were studied using human pancreas BxPC-3 cells. Deletion mutants of pTM-CAT plasmid revealed the presence of distal and proximal RA-responsive regions containing direct repeat with 4 spaces (DR4) and three of four Sp1 sites, respectively. Cotransfection of a pTM-CAT plasmid with expression plasmids of RA receptors (RARalpha, RARbeta, and RARgamma) augmented the promoter activity under the condition of lower retinoid X receptor-alpha (RXRalpha) expression, whereas the activity was greatly diminished when RXRalpha was highly expressed. An electrophoretic mobility shift assay with cDNA containing the DR4 indicated that heterodimers of RAR and RXRalpha interacted with the DR4 site, although the interaction gradually disappeared with the increase in the ratio of RXRalpha/RAR. On the other hand, Sp1 protein interacted especially with the tandem Sp1 site corresponding to the first and second Sp1 sequences of the four Sp1 sites in the proximal RA-responsive region. The binding of Sp1 to Sp1 sites was independent of RAR-RXR heterodimer but increased with the increase in Sp1 concentration in the presence of unknown factor(s) of reticulocyte lysate. Upon treatment of the cells with RA, time-dependent increases in the ratio of RARbeta to RXRalpha and the phosphorylated form of Sp1 were observed. We concluded that two genomic DNA regions, the DR4 site (-1531 to -1516) and the first and second Sp1-binding sites (-145 to -121), were involved in the RA-dependent augmentation of thrombomodulin gene expression through increased interactions of the two regions with heterodimer of RAR-RXRalpha and nuclear Sp1, respectively.

Binding Sites↗

PubMed2000

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.

Accidents, Traffic↗

PubMed2000

Sensory reconstruction in sensate radial forearm flap transfer.

The authors treated 14 patients (13 men and one woman), using a sensate radial forearm flap. Their ages at operation ranged from 27 to 67 years (mean: 52 years). Preoperative conditions were amputations in 10 cases, degloving injury in three, and crush injury in one. Reconstructive sites involved the thumb in nine cases, the mitten-like hand in two, the index finger in one, the ring and small finger in one, and the palm in one. In all cases, the radial forearm flap, including the lateral antebrachial cutaneous nerve, was harvested. Sensory evaluation was performed using the moving two-point discrimination test (m-2PD). Sensation in the mid-palmar area of 50 forearms was examined in 25 healthy adult volunteers as a control group. Follow-up periods ranged from 12 to 87 months (mean: 39.6 months). The mean m-2PD of the 14 sensory flaps was 13.2 mm, and the mean of 50 forearms in the control group was 18.08 mm. A statistically significant difference was demonstrated between the sensory flaps and the 50 forearms of the control group. The mean m-2PD was much more sensitive in the innervated radial forearm flaps than in the donor forearm. The results suggested that sensory return in the innervated flaps is influenced not by the donor nerve in the flaps, but by the recipient digital nerve.

Adult↗

PubMed1999

Choline acetyltransferase activity in collateral sprouting of peripheral nerve after surgical intervention: experimental study in rats.

The purpose of this study was to establish an assay of choline acetyltransferase (ChAT) activity to investigate the regeneration of injured peripheral nerve, repaired by end-to-end or end-to-side neurorrhaphy. Murine sciatic and peroneal nerves were exposed, and the peroneal nerve was transected at a site 5 mm from its ramification. For end-to-side neurorrhaphy, an epineurotomy producing a 5-x5-mm window was carried out on the tibial nerve, just above the level of gastrocnemius muscle ramification. The peroneal nerve stump was then sutured end-to-side to the tibial nerve window. For end-to-end neurorrhaphy, the peroneal stump was directly sutured end-to-end. ChAT activity was measured at a site distal to the peroneal stump at 1 to 3 months postoperatively, and the results were compared among four groups: 1) end-to-end neurorrhaphy group; 2) end-to-side neurorrhaphy group; 3) unrepaired group; and 4) positive controls. ChAT activity in the end-to-side neurorrhaphy yielded approximately two-thirds the value of the end-to-end neurorrhaphy, and more than half the value of positive controls at 3 months postoperatively. Histologic sections of the end-to-side and end-to-end sutured peroneal nerve demonstrated large numbers of myelinated axons and Schwann cells at the third postoperative month. All the results demonstrated that end-to-side neurorrhaphy is comparable to well-performed end-to-end neurorrhaphy, thus providing another option for surgical treatment of avulsion nerve injury and massive nerve defect.

Anastomosis, Surgical↗

PubMed1999

Free vascularized fibula grafts in surgery of the upper limb.

Twenty patients with intractable diseases in the upper extremity were treated using free vascularized fibula grafts. There were 13 men and seven women. Three patients had traumatic bone defects, five had post-traumatic nonunions, two had congenital pseudoarthroses, seven had defects after tumor resection, and three had other lesions. The reconstructed sites were the humerus in two patients, the radius and/or ulna in 17, and the metacarpal and phalangeal bones in one. The length of the bone defect ranged from 3 to 18 cm (mean: 8.4 cm). Follow-up periods ranged from 6 to 204 months. No patient required additional bone grafts. The mean period required to obtain radiographic bone union was 4.4 months. There were no cases with fractures of the grafted bone, but malunion occurred in four cases. The vascularized fibula graft is indicated in patients with large bone defects or intractable nonunions in the humerus, radius, and/or ulna.

Adolescent↗

PubMed1998

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.

Adolescent↗

PubMed1997

The potent inhibition of vapiprost, a novel thromboxane A2 receptor antagonist, on the secondary aggregation and ATP release of human platelets.

The inhibitory effects of vapiprost hydrochloride (vapiprost), a novel thromboxane A2 receptor antagonist, on platelet aggregation and ATP release were studied using platelet rich plasma (PRP) of humans, guinea pigs, rabbits and rats. In in vitro experiments with human platelet, vapiprost inhibited the aggregation and ATP release stimulated with U-46619, collagen or arachidonic acid (AA) at an IC50 of less than 2.1 x 10(-8) M. Vapiprost did not inhibit the primary aggregation or ATP release of human platelets stimulated with adenosine 5'-diphosphate (ADP), epinephrine (Epi) or platelet activating factor (PAF), but inhibited the secondary aggregation stimulated with those agonists at an IC50 of less than 1.3 x 10(-7) M. The sensitivity of platelets in various species of animals to vapiprost was in the following order: human > or = guinea pigs > rats > rabbits. In ex vivo experiments with guinea pigs which received a single oral dose of vapiprost, the agent demonstrated strong inhibition of ATP release from platelets stimulated with U-46619, collagen or AA at an ID50 of less than 25.8 micrograms/kg. These inhibitory effects were observed within 30 min and sustained for 24 h at a single dosage of 5 mg/kg of vapiprost. In AA-induced pulmonary infarction models of mice, the sudden death rates decreased significantly with the oral administration of 10 mg/kg or more of vapiprost. These results indicate that vapiprost effectively inhibits the secondary aggregation and ATP release of human platelets stimulated with various agonists, and that guinea pig and human platelets are similar in response to vapiprost. Furthermore, it was demonstrated in ex vivo experiments with guinea pigs that the inhibitory action of vapiprost appears rapidly and lasts for long periods.

Adenosine Triphosphate↗

PubMed1996

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.

Anti-Bacterial Agents↗

PubMed1994

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.

Cell Differentiation↗

PubMed1993

Different thrombomodulin induction in monocytic, macrophagic and neutrophilic cells differentiated from HL-60 cells.

Thrombomodulin (TM) antigen and its cofactor activity for thrombin-dependent protein C activation were not detected in the untreated HL-60 human promyelocytic cell line, but appeared in cells cultured with 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3: 10-1,000 nM) or phorbol 12-myristate 13-acetate (PMA: 0.1-10 nM) accompanied by an increase in TM mRNA levels. The induction of TM increased in parallel with the appearances of both nonspecific esterase activity, a typical marker of monocyte/macrophage lineages, and phagocytic activity. The TM antigen level induced in 1 alpha,25(OH)2D3-treated cells was 8 times higher than that in PMA-treated cells. Trace amounts of TM antigen were induced in neutrophilic cells differentiated from HL-60 by treatment with retinoic acid. These results indicated that different levels of TM were induced in monocytic, macrophagic and neutrophilic cells differentiated from HL-60 cells.

Calcitriol↗

PubMed1993

[Effects of NC-1100, a calcium channel blocker, on experimental cerebral ischemia/anoxia in rodents].

The anti-ischemic and anoxic effects of NC-1100, a piperazine type calcium channel blocker, were investigated in various cerebral ischemia and anoxia models in mice, gerbils and guinea pigs. Minimal effective doses of NC-1100 were 8 mg/kg, i.p. and 30 mg/kg, p.o. for KCN-induced anoxia; 16 mg/kg, i.p. for decapitation-induced gasping; 30 mg/kg, i.p. for cerebral ischemia induced by occlusion of bilateral carotid arteries in gerbils; and 10 microM for the in vitro ischemic model in hippocampal slices. Moreover, NC-1100 attenuated the disturbance of cerebral energy metabolism induced by decapitation in mice. These results suggest that NC-1100 has a cerebral protective effect, and that attributable to its ability to improve the cerebral energy metabolism disturbance.

Animals↗

PubMed1992

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.

Cells, Cultured↗

PubMed1992

Retinoic acid stimulates expression of thrombomodulin, a cell surface anticoagulant glycoprotein, on human endothelial cells. Differences between up-regulation of thrombomodulin by retinoic acid and cyclic AMP.

Thrombomodulin (TM) is a surface protein on endothelial cells, and represents one of the most valuable regulatory factors in the anticoagulant system. In this paper, we demonstrate that retinoic acid (RA) causes an increase in TM antigen on human umbilical vein endothelial cells (HUVECs) in vitro. The effect of RA on the surface TM level of HUVECs was dose-dependent in the range from 0.01 to 10 microM-RA. Antigen levels began to increase 3 h after addition of 10 microM-RA, and plateaued at a maximum level of approx. 2.5 times that of the untreated control at 24 h. TM levels remained at a maximum for a further 12 h, and then gradually decreased. The effects of RA on cell surface TM activity and antigen levels were parallel in all experiments. TM expression was also increased by treatment with 10 microM-retinal or 10 microM-retinol for 24 h, though the increases were approx. 70% and 30% respectively of that produced by 10 microM-RA. Pretreatment of HUVECs with cycloheximide inhibited the effect of RA. When HUVECs were incubated with both 10 microM-RA and 5 mM-8-bromo cyclic AMP (or 1 mM-3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor), the increase in TM antigen was greater than that observed with either compound alone. Northern blot analysis showed that treatment of HUVECs with 8-bromo cyclic AMP, RA or RA plus 8-bromo cyclic AMP increased TM mRNA levels by 2.2-, 4.5- and 5.5-fold respectively compared with the untreated control. Furthermore, no significant difference in cellular cyclic AMP levels was observed between RA-treated and control cells. These results indicate that the expression of TM is not only controlled by the intracellular cyclic AMP level but is also affected by RA, and suggest that RA-induced up-regulation of TM on HUVECs is independent of cyclic AMP regulation.

8-Bromo Cyclic Adenosine Monophosphate↗