Expression and activity of thrombomodulin in human gingival epithelium: in vivo and in vitro studies.

Epidermal keratinocytes thrombomodulin (TM) has been shown to regulate thrombin at sites of cutaneous injury in addition to a role for epidermal differentiation. TM, a major anticoagulant proteoglycan of the endothelial cell membrane, is a thrombin receptor that acts as a co-factor for protein C activation. Thrombin has pro-inflammatory effects for periodontitis. However, little is known about TM in gingival tissue with periodontitis. We used immunohistochemistry to examine expression of TM in gingival epithelium from patients with periodontitis. In vitro, we observed TM expression at varying Ca2+ concentrations by confocal laser scanning microscopy, examined the expression of TM mRNA and tested TM co-factor activity. Furthermore, we measured TM concentration in gingival crevicular fluid (GCF) from 11 severe adult cases of periodontitis using enzyme-linked immunosorbent assay. Immunoreactive TM was present in gingival epithelium and junctional epithelium, and was reduced in inflamed gingival epithelium compared to healthy gingival epithelium. Ultrastructurally, TM, including microvilli, was observed on the cell membrane. TM localization in cells cultured in 0.09 mM Ca2+ differed from that in cells exposed to 1.2 mM Ca2+. Northern analysis demonstrated TM mRNA in gingival keratinocytes more than in human umbilical vein endothelial cells (HUVEC). Gingival keratinocytes also facilitated protein C activation by thrombin, although less strongly than HUVEC. TM in GCF at sites with bleeding on probing in patients was significantly elevated (p < 0.001, Student's t-test). TM in gingival epithelium may regulate thrombin activity at sites of coagulation and inflammation with periodontal disease, although inflammation may impair this regulation of thrombin.

Transvaginal color Doppler ultrasonic characterization of benign and malignant ovarian cystic teratomas and comparison with serum squamous cell carcinoma antigen.

BACKGROUND: The preoperative diagnosis of squamous cell carcinoma (SCC) arising in mature cystic teratoma of the ovary remains difficult. The purpose of this study is to examine the usefulness of transvaginal color Doppler ultrasound (TV-CDU) in differentiating malignant (SCC) from benign cystic teratoma of the ovary. METHODS: Eighty-eight patients with an ovarian tumor showing gray scale sonographic appearances of mature cystic teratoma were preoperatively evaluated for the presence or absence of intratumoral blood flow by TV-CDU. The blood flow characteristics of the tumor vessels were analyzed using the resistance index (RI), pulsatility index (PI), and peak systolic velocity (PSV). The serum levels of SCC antigen were also randomly examined preoperatively in 50 patients. RESULTS: Intratumoral blood flow was significantly detected in malignant teratomas (SCCs) (80.0%; 4 of 5) compared with benign teratomas (20.5%; 17 of 83) (P < 0.01). All malignant teratomas with intratumoral blood flow showed both RI less than 0.4 and PI less than 0.6, whereas no benign teratomas showed any such value except for 1 case with struma ovarii. In addition, both the mean RI and the mean PI values in the tumor vessels were significantly lower in the malignant teratomas (RI: 0.31 +/- 0.07; PI: 0.40 +/- 0.16) than in the benign teratomas (RI: 0.62 +/- 0.13; PI: 1.06 +/- 0.44) (P < 0.001). However, the mean PSV value of the malignant teratomas (PSV: 20.6 +/- 8.33) was not significantly different from the benign teratomas (PSV: 18.1 +/- 9.9). Elevation of serum SCC was found in 4 of 5 patients (80%) with malignant teratomas, whereas the elevation was found in 11 of 45 patients (24.4%) with benign teratomas (P < 0.05). The diagnostic accuracy using the RI (cutoff value 0.4) as well as the PI (cutoff value 0.6) was thus 95.2%, which was significantly superior to that obtained by using the serum SCC (76%) (cutoff value, 1.5 ng/mL). CONCLUSIONS: Evaluating the presence or absence of intratumoral blood flow, together with blood flow resistance, in tumor vessels using TV-CDU thus may be more useful to differentiate malignant (SCC) from benign cystic teratomas of the ovary than by measuring serum SCC levels.

Expression of Thrombomodulin in the epithelium of the urinary bladder: a possible source of urinary thrombomodulin.

Thrombomodulin (TM) is an endothelial thrombin receptor which acts as a natural anticoagulant through inactivation of the procoagulant activity of thromin. In the present study, we demonstrated that TM is expressed on the urinary bladder epithelium. The cell line BOY established from human transitional cell carcinoma of the urinary bladder also expressed the TM message (3.7kb). These cells express an 85 kDa TM protein which is identical in size to that of MEG-01, a human megakaryoblastic cell line, reported previously. We also ascertained the expression and localization of TM in the human urinary bladder by immunohistochemical staining. TM was localized in the membranes of the transitional epithelium and the cytoplasmic region of umbrella cells. The expression of TM in the transitional epithelium increased with worsening pathological status of cystitis. Based on these results, we concluded that TM is expressed in the urinary bladder. Further, soluble TM in urine may be partially derived from the urinary bladder. At present, the physiological significance of TM expression in the urinary bladder remains to be elucidated.

The blood flow characteristics in borderline ovarian tumors based on both color Doppler ultrasound and histopathological analyses.

To clarify the tumor behavior in borderline ovarian tumors, we examined the characteristics of neovascularization in these tumors by using a transvaginal color Doppler ultrasound (TV-CDU). Twelve patients with borderline ovarian tumors were preoperatively evaluated for the characteristics of intratumoral blood flow by TV-CDU, using both the resistance index (RI) and pulsatility index (PI). As a control group, 100 patients with benign ovarian tumors and 31 patients with malignant ovarian tumors were also examined by TV-CDU. An intratumoral blood flow was significantly detected in both borderline (91.6%; 11/12) and malignant ovarian tumors (90.3%; 28/31), but not in benign ovarian tumors (53%; 53/100) (P < 0.01). In addition, both the mean RI and mean PI values were significantly lower in the borderline (RI; 0.45, PI; 0.67) and malignant ovarian tumors (RI; 0.39, PI; 0.58) than those in the benign ovarian tumors (RI; 0.61, PI; 1.05) (P < 0.01). In mucinous tumors, the borderline tumors showed a significantly high intratumoral vascularity (P < 0. 01) and both borderline and malignant tumors significantly demonstrated a low-resistance blood flow (P < 0.01), in comparison to those of the benign tumors. Mucinous borderline tumors of the intestinal type also tended to have a lower RI as well as a lower PI value than müllerian type. Regarding neovascularization as represented by intratumoral blood flow characteristics, this study thus suggests that a close relationship exists in the tumor behavior between borderline and malignant ovarian tumors, especially in mucinous epithelial tumors.

Myocardial infarction induces expression of midkine, a heparin-binding growth factor with reparative activity.

We examined midkine (MK) expression in the rat heart upon experimental myocardial infarction. Immunohistochemical staining revealed, 6 hours after ligation of the left anterior descending coronary artery, strong MK immunoreactivity in myocytes and endothelial cells of a non-infarcted cardiac region. The myocytes of the infarcted cardiac region destined for death showed only a little immunoreactivity. Northern blot analysis suggested that the increased immunoreactivity was due to increased MK synthesis. The induced MK expression is likely to mimic the expression during embryogenesis: MK was strong lye-expressed in the myocytes of embryonic heart, and the expression decreased during embryogenesis.

Identification of the domain of alpha-catenin involved in its association with beta-catenin and plakoglobin (gamma-catenin).

alpha-Catenin is a 102-kDa protein exhibiting homology to vincuin, and it forms complexes with cadherins or the tumor-suppressor gene product adenomatous polyposis coli through binding to beta-catenin or plakoglobin (gamma-catenin). The incorporation of alpha-catenin into the cadherin-catenin complexes is a prerequisite for expression of the cell-adhesive activity of cadherins. Using an in vitro assay system involving bacterially expressed proteins, we localized a region in alpha-catenin required for molecular interaction with beta-catenin and plakoglobin. Analysis of various truncated alpha-catenin molecules revealed that amino-terminal residues 48-163 are able to bind to beta-catenin and plakoglobin. Consistent with the observation that beta-catenin and plakoglobin bind to the same region of alpha-catenin, beta-catenin competed with the binding of plakoglobin to alpha-catenin and vice versa. Under the conditions used, beta-catenin bound to alpha-catenin with higher affinity than did plakoglobin. Scatchard analysis indicated that the affinity of the interaction between alpha-catenin and beta-catenin or that between alpha-catenin and plakoglobin was moderately strong (Kd = 3. 8 x 10(-8) and 7.7 x 10(-8), respectively). When transfected into L cells expressing E-cadherin, the amino-terminal region of alpha-catenin (from residue 1 to 226) formed complexes with beta-catenin supporting the in vitro binding experiment results.

A transgenic mouse line with alpha-1,3/4-fucosyl-transferase cDNA: production and characteristics.

cDNA of human alpha-1,3/4-fucosyltransferase (Fuc-TIII) was placed under the control of the chicken beta-actin promoter and cytomegalovirus enhancer, then introduced into male pronuclei of fertilized mouse eggs. A transgenic mouse line thus obtained exhibited enhanced expression of Lex (4C9) antigen in endothelial cells located in the glomerulus, sinusoidal capillaries of the liver and capillaries of the heart. Furthermore, in the transgenic mice, sialyl dimeric Lex (FH6) and sialyl Lea (2D3) antigens were strongly expressed in the glomerular endothelial cells.

Enhanced expression of type I receptors for bone morphogenetic proteins during bone formation.

Type I receptors for bone morphogenetic proteins (BMPs), i.e., BMPR-IA and BMPR-IB, are transmembrane serine/threonine kinases, that bind osteogenic protein-1 (OP-1, also termed BMP-7) and BMP-4. Using antibodies specific to BMPR-IA and -IB, we have studied the expression of BMP type I receptors in the bone formation process during embryonic development and fracture healing. In the mouse embryo, both BMPR-IA and -IB were expressed in condensing mesenchymal cells at 13.5 days post coitum (p.c.). At 15.5 days p.c., expression of BMPR-IB, but not of BMPR-IA, was observed in the cells in perichondrium of developing cartilage. At 17.5 and 19.5 days p.c., expression of both receptors was observed in chondrocytes and in osteoblasts. In normal rat adult bone, expression of BMPR-IA, but not of BMPR-IB, was observed in osteoblasts in the periosteum. Three days after the femoral fracture, expression of BMPR-IA and -IB was up-regulated in cells at the proliferating osteogenic layer of the periosteum. On day 7, both receptors were found in fibroblast-like spindle cells and chondrocytes in the endochondral ossification sites, and osteoblasts in the newly formed trabecular bone. Expression of BMPR-IA was higher than that BMPR-IB in osteogenic layer on day 3 and in osteoblasts in the trabecular bone on day 7. On day 14, expression of BMP type I receptors was observed at similar sites, albeit with lower expression levels than were observed on day 7. The present data suggest that expression of BMP type I receptors is up-regulated during bone formation, and that they may play important roles in bone morphogenesis.

Blood coagulation equilibrium in rat liver microcirculation as evaluated by endothelial cell thrombomodulin and macrophage tissue factor.

The regulatory mechanisms of microcirculation might differ in the liver from other organs, because macrophages are resident in the hepatic sinusoids and sinusoidal endothelial cells are unique in shape and function. Thrombomodulin expression in endothelial cells and tissue factor activity in isolated macrophages were studied in the liver and lung of rats. In normal rats, the thrombomodulin expression was minimal in hepatic sinusoids, but prominent in pulmonary capillaries, while the tissue factor activity in the presence of endotoxin was higher in pulmonary macrophages than in Kupffer cells, although the levels in the absence of endotoxin were comparable in both cells. The tissue factor activity in hepatic macrophages was increased after priming of the cells with Corynebacterium parvum or after induction of liver necrosis or cirrhosis with carbon tetrachloride. In the necrotic or cirrhotic liver, increased thrombomodulin expression was seen along capillaries extending in necrotic areas and regenerating nodules, but this increase was minimal in the Corynebacterium parvum-treated rat liver. Blood coagulation equilibrium in microcirculation regulated by endothelial cells and macrophages may differ between the liver and lung. Such equilibrium in the liver may vary depending on pathological status.

Expression of thrombomodulin in esophageal squamous cell carcinoma and its relationship to lymph node metastasis.

Thrombomodulin (TM) is thrombin receptor that was identified originally on the endothelium and acts as a natural anticoagulant. However, we reported previously that TM was also expressed in the squamous epithelium mainly at the intercellular bridges. In this study, we examined TM expression in the primary lesions of 106 patients with esophageal squamous cell carcinomas and in the lymph node metastatic lesions of 59 patients using immunohistochemical methods. The carcinoma tissues expressed TM mainly at the cell-cell boundaries and in the cytoplasm. When TM expression was compared between the primary and metastatic lesions in the 59 patients who had lymph node metastasis, 41 (69%) showed decreased TM expression, 18 (31%) showed no change, and none (0%) showed an increase in the metastatic lesions. Wilcoxon's signed-rank test indicated that tumor cells that were positive for TM expression were significantly rarer in the metastatic lesions than in the primary tumors (P < 0.0001). This result indicates that the decrease in TM expression is associated with metastasis of the carcinoma cells. This phenomenon is very similar to that of E-cadherin, although the structures of both molecules are quite different. The reduction of TM expression seems to play an important role in the metastatic process of esophageal cancer.

Midkine (MK) expression in extraembryonic tissues, amniotic fluid, and cerebrospinal fluid during mouse embryogenesis.

Midkine (MK) and pleiotrophin (PTN) constitute a new family of heparin-binding growth factors. Extraembryonic membranes and the placenta of the mouse expressed MK mRNA at 11.5 days gestation. While the MK mRNA level in extraembryonic membranes decreased during embryogenesis, that in the placenta remained unchanged. Immunohistochemical studies showed that Mk was located in the yolk sac and in the amnion at 11.5 days gestation. PTN mRNA expression was weak in extraembryonic membranes and was scarcely detectable in the placenta. Western blot analysis revealed the presence of MK in amniotic fluid and cerebrospinal fluid, in amounts of more than 1 microgram/ml, raising the possibility that MK delivered by these fluids participates in the regulation of organogenesis. Transport of MK from the site of its synthesis appears to also occur in the adult kidney, since MK mRNA and the MK protein are localized in different regions of the kidney.

Identification of nucleolin as a binding protein for midkine (MK) and heparin-binding growth associated molecule (HB-GAM).

Midkine (MK) is a heparin-binding growth/differentiation factor with a molecular weight of 13 kDa, and is structurally unrelated to fibroblast growth factors (FGF). We studied MK-binding proteins in order to clarify the action mechanism of MK. A 100-kDa protein was identified in PYS-2, 3T3, and L cells as an MK-binding protein by a ligand blot experiment. This MK-binding protein was purified by affinity chromatography on an MK-agarose column followed by SDS polyacrylamide gel electrophoresis. Sequence determination of N-terminal 23 amino acid residues revealed that the MK-binding protein was nucleolin, a major nucleolar protein, which functions as a shuttle protein between the nucleus and cytoplasm and is located also on the cell surface. Heparin-binding growth associated molecule (HB-GAM), which has 50% sequence identity with MK, fused to maltose-binding protein also bound to nucleolin. On the other hand, basic FGF (bFGF) scarcely bound to nucleolin in the absence of heparin, while both MK and bFGF bound weakly to nucleolin in the presence of heparin. Nuclear localization of MK was shown in hemangioma cells by immunohistochemical staining. These findings supported the hypothesis that parts of the MK and HB-GAM are translocated to the nucleus after binding with nucleolin.

The midkine (MK) family of growth/differentiation factors: structure of an MK-related sequence in a pseudogene and evolutionary relationships among members of the MK family.

Midkine (MK) is a novel heparin-binding growth/differentiation factor coded by a retinoic acid-responsive gene. MK cDNA probe reacts with two bands, a 4 kb one and a 3 kb one, upon Southern blot analysis of Hin dIII fragments of mouse genomic DNA: the midkine gene (Mdk) is on the 4 kb fragment. Sequence analysis of the 3 kb fragment revealed that it has an Mdk-related sequence (Mdk-rs) highly homologous to MK cDNA, three mouse Aluequivalent repeats and seven A+T-rich segments. The Mdk-rs carried an inserted microsatellite DNA, is flanked by imperfect direct repeats observed in many retroposons, and lacks introns. Interspecific hybrid analysis revealed that Mdk-rs is located on chromosome 11, while Mdk is known to be on chromosome 2. The evolutional velocity of Mdk-rs was calculated to be 11 times higher than that of mouse Mdk. These features suggest that Mdk-rs is a processed pseudogene generated in the mouse genome. The 3 kb fragment with Mdk-rs, which is rich in inserted DNA sequences probably due to the presence of A+T-rich segments, may be a hot spot for amplification and evolution of genomic DNA. Mdk-rs was estimated to have been generated about 19.1 million years ago. A chicken protein retinoic acid-induced, heparin-binding protein (RIHB), is highly homologous to MK, and its divergence from human MK was estimated to have occurred about 250 million years ago, suggesting that RIHB is the chicken homology of MK. Thus, so far there are only two established protein members, MK and heparin-binding, growth-associated molecule (HB-GAM)/pleiotrophin (PTN) in the MK family.

Transgenic mouse lines with ectopic expression of alpha-1,3-galactosyltransferase: production and characteristics.

The cDNA of murine alpha-1,3-galactosyltransferase was placed under the control of the beta-actin promoter and cytomegalovirus enhancer, then introduced into male pronuclei of fertilized mouse eggs. The three transgenic mouse lines obtained were analysed for the expression of the transferase by staining with Griffonia simplicifolia agglutinin I-B4 (GSI-B4), which is alpha-galactosyl specific. Compared with wild-type mice, all lines of transgenic mice expressed GSI-B4 binding sites more intensely in the renal tubular brush border and lung alveolar epithelium, and newly expressed them in the photoreceptor outer segments, goblet cells of the small intestine and around spermatogonia. GSI-B4 binding sites were also detected in the liver of some transgenic mice. Even though the introduced enzyme gene was expressed in embryos, it did not severely hinder embryogenesis. The transgenic mice tended to secrete more proteins in the urine than the wild type. Furthermore, low body weights, partial damage to hair growth and early death occurred more frequently in the transgenic mice.

A new family of heparin binding growth/differentiation factors: differential expression of the midkine (MK) and HB-GAM genes during mouse development.

MK (midkine) and HB-GAM (heparin-binding growth-associated molecule) constitute a new family of heparin-binding growth differentiation factors. The modes of expression of MK and HB-GAM during mouse development were quantitatively examined by mRNA hybridization. The following three distinct patterns of expression were observed in the brain/head region. On the 11th-13th days of gestation, MK was intensely, but HB-GAM relatively weakly expressed; on the 15th-19th days, both MK and HB-GAM expression became weaker; and in the neonatal period, HB-GAM was intensely expressed and MK expression increased slightly. The level of HB-GAM expression was lower than that of MK in the whole embryo on the 11th to 13th days of gestation. HB-GAM mRNA was detected in the kidney of newborn and young mice, where MK was more highly expressed. The identity of the weakly expressed MK and HB-GAM signals was confirmed by means of the polymerase chain reaction in the neonatal brain (MK), the head of 13-day embryos (HB-GAM), and the kidney of 7-day-old mice (HB-GAM). In conclusion, MK and HB-GAM are frequently co-expressed in the same cells and anatomic regions of the fetus or new born mouse, while their modes of expression differ.