Transition of pathophysiological significance of plasminogen activator inhibitor-From a chief player in antiinflammation, antifibrinolysis to that in the development of insulin resistance.

In the early phase of research, plasminogen activator inhibitor (PAI) was regarded as a negative regulator of fibrinolytic system, but the later study clarified that the changes in PAI level is closely related to risk factors of various pathologic processes of the lifestyle-related diseases. It is accepted that PAI-1 is a risk factor of the cardiovascular event in lifestyle-related diseases by recent researches analyzing the detailed function of PAI-1. In this review paper, we described the transition of pathophysiological significance of PAI based on many research papers especially from ours, which clarified the mechanism on protein expression of PAI, especially PAI-1.

RNAi targeting embryonic myosin heavy chain isoform inhibited bound thrombin-induced migration of vascular smooth muscle cells.

To investigate the effect of bound thrombin, a complex of alpha-thrombin with fibrin fragments derived from clots, on proliferation and migration of cultured rabbit vascular smooth muscle cells, cell proliferation was measured by WST-1 reagent and migration was evaluated by counting migrated cells through pores of cell culture insert (8 mum size) after 48-hour treatment with bound thrombin (10 U/ml). To examine the role of an embryonic myosin heavy chain isoform (SMemb) in these effects by bound thrombin, the cells were subsequently treated for 48 h with an siRNA expression vector (ORF-2/pSilencer) directed against the open reading frame of SMemb mRNA. SMemb and plasminogen activator inhibitor-1 mRNA expressions were measured by Northern blot analysis. Bound thrombin significantly increased SMemb mRNA expression by 1.4 +/- 0.01-fold and significantly increased plasminogen activator inhibitor-1 mRNA expression by 2.65 +/- 0.69-fold (p < 0.01 vs. PBS treatment for each), which were abolished by treatment with ORF-2/pSilencer. Although bound thrombin had no effect on cell proliferation, bound thrombin significantly increased migration by 1.93 +/- 0.20-fold (p < 0.05). ORF-2/pSilencer treatment significantly reduced the bound thrombin-stimulated migration activity by 1.28 +/- 0.15-fold (p < 0.05). Thus, SMemb plays an important role in bound thrombin-induced cell migration activity of cultured vascular smooth muscle cells.

Cloning of habutobin cDNA and antithrombotic activity of recombinant protein.

The habutobin cDNA was cloned from total RNA extracted from venom glands of Trimeresurus flavoviridis (the habu snake). The conceptual translation of 1539 bp of habutobin cDNA consists of 236 amino acids and its molecular weight is 25.7 kDa. Histidine (His)-tagged recombinant habutobin fusion protein, pET-r-habutobin and AcNPV-r-habutobin, was purified by bacterial system and baculoviral system, respectively. After refolding pET-r-habutobin, there were two protein bands at about 32 kDa and 65 kDa, indicating that habutobin might be produced as a monomer protein and processed to form two concatenated protein. Purified AcNPV-r-habutobin dose-dependently increased fibrin forming activity and inhibited collagen-induced aggregation of rabbit washed platelets. Thus, AcNPV-r-habutobin produced by baculoviral system is very useful for study on structure-function relationship, which is necessary for developing an antithrombotic drug from habutobin.

Inhibition of IgE-mediated phosphorylation of FcepsilonRIgamma protein by antiallergic drugs in rat basophilic leukemia (RBL-2H3) cells: a novel action of antiallergic drugs.

We examined the effect of antiallergic drugs, azelastine and epinastine, on the expression of FcepsilonRIalpha, beta, and gamma chains and phosphorylation of the gamma chains in rat basophilic leukemia (RBL-2H3) cells. The cells were cultured for 24 h with IgE treatment in the presence of azelastine or epinastine at the concentration of 10(-5) M. The FcepsilonRIalpha mRNA expression was determined by northern blot analysis. The protein level of FcepsilonRI expressed on the plasma membrane was examined following IgE treatment by immunoprecipitation with anti-IgE light chain, followed by western blot analysis with anti-gamma chain of FcR. Azelastine and epinastine had no effect on the FcepsilonRIalpha, beta and gamma mRNA levels. Although the amount of gamma chain assembled into IgE-bound FcepsilonRI was not changed by treatment with azelastine nor epinastine, phosphorylation levels of gamma chains of IgE-bound FcepsilonRI were inhibited by azelastine. The inhibitory effect of azelastine on the IgE-mediated expression of FcepsilonRIgamma protein is not due to their inhibition of mRNA and protein expression, but due to abrogating phosphorylation of the gamma chains, which is important for initiation of FcepsilonRI signaling cascade elicited by IgE interaction.

RNA interference targeting embryonic myosin heavy chain isoform inhibited mRNA expressions of phenotype markers in rabbit cultured vascular smooth muscle cells.

To investigate whether the knockdown of SMemb gene expression induces phenotypic modulation of vascular smooth muscle (VSM) cells toward a contractile type, we constructed a siRNA targeting the 3' untranslated region (UTR) of SMemb gene (SMemb-siRNA). The SMemb-siRNA was introduced into cultured rabbit VSM cells for 48 h at 37 degrees C by the lipofection method. The mRNA expressions were estimated by comparative reverse transcription-polymerase chain reaction (RT-PCR). SMemb-siRNA significantly decreased the ratio of SMemb to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression in a dose-dependent manner (P < 0.01): 0 nM, 0.90 +/- 0.08; 100 nM, 0.43 +/- 0.07. Immunofluorescence and immunoblot analyses demonstrated that SMemb-siRNA markedly decreased SMemb protein expression to 56% +/- 7.8% (P < 0.01). Other MHC isoform (SM1 and SM2) mRNA expressions were not changed. The relative mRNA expressions of other phenotype markers (plasminogen activator inhibitor (PAI)-1 and beta-actin) were significantly decreased by SMemb-siRNA to 71% +/- 7.5% and 61% +/- 7.5%, respectively (P < 0.01). Expression of smooth muscle (SM) alpha-actin protein and cell proliferation was not changed by SMemb-siRNA. Thus, SMemb gene might be involved in the transcription of PAI-1 and beta-actin, but not involved in SM alpha-actin and cell proliferation in cultured VSM.

Involvement of Epstein-Barr virus (EBV)-encoded latent membrane protein (LMP)-1 in the potentiation of C epsilon mRNA expression in human tonsil-derived cells.

The aim of our study was to investigate a possible correlation between Epstein-Barr virus (EBV) infection status, including its latent gene expression, and expression of allergy-related genes in human tonsil-derived cells. In the tonsil-derived cells from the patients undergoing routine tonsillectomies for palatine tonsil hypertrophy or tonsillar focal infection, the presence of EBV DNA and mRNA expressions of latent membrane protein (LMP)-1, C epsilon chain, and activation-induced cytidine deaminase (AID) were detected by PCR and RT-PCR analysis, respectively. Of all the 12 patients, PCR products amplified from EBV DNA BamHI W fragment were detected in the tonsils from the 10 patients (83.3%). LMP1 mRNA expressions were confirmed in the six patients (50%). Both LMP1 mRNA expressions and EBV DNA were detected in the five patients. EBV DNA, but not LMP1 mRNA expression, was detected in the five patients. LMP1 mRNA expression, but not EBV DNA, was detected in one patient. In one patient, neither EBV DNA nor LMP1 mRNA expression was confirmed. C epsilon mRNA expressions were confirmed in all the 12 patients along with AID mRNA expressions. The degree of C epsilon mRNA expression, however, varied with the patients. The Fisher's exact probability test revealed a statistically significant correlation between LMP1 and C epsilon gene expressions, indicating that C epsilon mRNA expression level was significantly higher in the LMP1 positive samples than in the negative samples. On the other hand, there was no significant correlation between AID and LMP1 mRNA expressions. Thus, EBV infection is a notable factor capable of exacerbating allergic inflammation.

Elevation of intracellular cAMP up-regulated thrombomodulin mRNA in cultured vascular endothelial cells derived from spontaneous type-II diabetes mellitus model rat.

To investigate whether antihemostatic function of vascular endothelial cells (VECs) is changed in type-II diabetic model rats, the mRNA expressions of tissue-type and urokinase-type plasminogen activator (t-PA and u-PA), thrombomodulin (TM), PA inhibitor type-1 (PAI-1), and phosphodiesterases (type 3A, 3B, and 4D PDEs) were quantitated by the method of comparative reverse transcriptase-polymerase chain reaction (RT-PCR). VECs from type-II diabetic model Otsuka Long-Evans Tokushima Fatty (OLETF) rats and from its normal counterpart (LETO) rats were cultured for 24 h with dibutyryl adenosine 3',5'-cyclic monophosphate (db-cAMP) or a type-3 PDE inhibitor, cilostazol. Intracellular cAMP concentration was determined by the chemiluminescent enzyme-linked immunosorbent assay (ELISA) system. In cultured VECs from OLETF rats, the basal mRNA expressions of u-PA and TM were significantly decreased as compared to those in cultured VECs from LETO rats. TM mRNA expression in cultured VECs from OLETF rats was increased 2.1-fold at 24 h after treatment with db-cAMP (3 mmol/L). Basal mRNA expressions of type 3A, 3B, and 4D PDEs were significantly higher in VECs from OLETF rats than those from LETO rats. After treatment with cilostazol (30 micromol/L), intracellular cAMP was significantly increased at 60 min and TM mRNA expression was increased 1.5-fold at 24 h. Therefore, elevation of intracellular cAMP by db-cAMP or cilostazol up-regulated TM mRNA expression in cultured VECs from OLETF rats.

Antiallergic drugs, azelastine hydrochloride and epinastine hydrochloride, inhibit ongoing IgE secretion of rat IgE-producing hybridoma FE-3 cells.

We asked whether or not antiallergic drugs, azelastine hydrochloride and epinastine hydrochloride, inhibit IgE secretion from IgE-producing hybridoma FE-3 cells. FE-3 cells were cultured in the presence of azelastine or epinastine for 24 h, washed in phosphate-buffered saline , and then recultured in the medium in the absence of the antiallergic drugs. IgE levels in the cultured medium as well as those in the cytoplasm of FE-3 cells were measured by enzyme-linked immunosorbent assay. mRNA levels of Cepsilon, activation-induced cytidine deaminase (AID), XBP-1, and Bip were estimated by northern blot or reverse transcriptase polymerase chain reaction analysis. The activities of nuclear factor-kappa B (NF-kappaB) were analyzed by electrophoretic mobility shift assay (EMSA). Phosphorylation of I kappa B alpha (IkappaBalpha) was analyzed by immunoprecipitation followed by western blot analysis. IgE levels in the cultured medium and in the microsome fraction were lower on the treatment with 10(-5) M azelastine or epinastine than those on the treatment with vehicle. Cepsilon and AID mRNA levels were lower on the treatment with 10(-5) M azelastine than those on the treatment with vehicle, but were not decreased on the treatment with 10(-5) M epinastine. XBP-1 and Bip mRNA levels were not altered following treatment of the antiallergic drugs. Azelastine at 10(-5) M, but not epinastine, reduced DNA binding activity of NF-kappaB and also diminished IkappaBalpha phosphorylation, leading to sustaining IkappaBalpha protein levels. These findings suggest that azelastine exerts its inhibitory effect on the IgE secretion from FE-3 cells through the inhibition of Cepsilon mRNA expression, and that the inhibitory effect of epinastine is, at least in part, due to suppression of IgE synthesis at the post-transcriptional level.

Modification of Cepsilon mRNA expression by EBV-encoded latent membrane protein 1.

To evaluate the effect of expression of latent membrane protein (LMP) 1 encoded by Epstein-Barr virus (EBV) on Cepsilon mRNA expression, mRNA levels were examined by RT-PCR or Northern blot analysis upon transient transfection of LMP1 in the splenocytes derived from Brown-Norway rats with or without immunization with 2,4-dinitrophenyl-conjugated Ascaris suum antigen. Splenocytes were transfected with LMP1 expression vector, pSG5-LMP1, using lipofection method. Cepsilon mRNA levels were considerably increased by transfection with pSG5-LMP1 in the splenocytes derived from the nonimmunized rats; however, Cepsilon mRNA levels were decreased in the splenocytes derived from the immunized rats. Cepsilon mRNA expression in IgE-producing cells are modulated by LMP1, which might depend on the differentiation status of B cells upon exposure to allergen.

Effect of sodium channel blocker, pilsicainide hydrochloride, on net inward current of atrial myocytes in thyroid hormone toxicosis rats.

To investigate effect of pilsicainide hydrochloride (pilsicainide) on electrocardiogram (ECG) signals and action potentials (APs) of atrial myocytes, levo-thyroxine (T4, 500 microg/kg body weight) was daily injected into peritoneal cavity of Sprague-Dawley rats for 14 days. T4-treatment significantly shortened RR interval, P wave, and QRS complex durations on ECG. Although pilsicainide did not affect the heart rate, P wave and corrected QT interval (QTc) was increased in T4-treated rats. AP recordings revealed that AP durations at 20%, 50%, and 90% repolarization were significantly shortened and maximal rate of rise (Max dV/dt) was significantly increased in T4-treated rat atrial cells. Pilsicainide significantly decreased AP amplitude (APA) and Max dV/dt in both control and T4-treated rat atrial cells. Concentration-inhibition study demonstrated that pilsicainide significantly inhibited net inward current of T4-treated rats at lower concentration (IC50 of 29.2 microg/mL) than that of control rats (133 microg/mL). In conclusion, pilsicainide could decrease the conduction velocity in T4-treated rat atrium by decreasing the Max dV/dt and net inward current, which could be a possible treatment of thyrotoxicosis-induced arrhythmia.

Electrophysiologic characteristics of atrial myocytes in levo-thyroxine-treated rats.

To investigate whether thyroid hormone modulates electrical properties of atrial myocytes, electrocardiogram (ECG), action potentials (APs), and ionic currents were measured. Male Sprague-Dawley rats were randomly divided into control and levo-thyroxine (T4)-treated groups at 6 weeks of age. Levo-thyroxine (500 microg/kg of body weight) was injected daily into the peritoneal cavity for 14 days (T4-treated rats) and the same volume of saline was injected in control rats daily. ECG signals were recorded using apex-base leads. APs, voltage-dependent Na+ and L-type Ca2+ channel current (I(Na) and I(Ca(L))), inwardly rectifying K+ channel current (I(K1)), transient outward K+ channel current (I(to)), and delayed rectifier K+ channel current (I(K(delay))) were measured using patch-clamp techniques. T4 treatment significantly changed electrical properties in rat atrial myocytes, including (1) the increase in heart rate, (2) the increase in cell size, (3) the shortening of action potential duration (APD), (4) the increase in cell membrane capacitance (C(m)), and (5) the decrease in input resistance (R(in)). Although the current densities of I(Na) and I(K1) in T4-treated atrial myocytes did not differ from those in control cells, I(Ca(L)) was significantly decreased and I(K(delay)) was significantly increased in T4-treated rats. Thus, thyrotoxicosis could induce the shortening of APD by alterations in current density of both I(Ca(L)) and I(K(delay)) in rat atrial myocytes.

Argatroban, specific thrombin inhibitor, induced phenotype change of cultured rabbit vascular smooth muscle cells.

To investigate whether argatroban ((2R,4R)-4-methyl-1-[N(2)-((RS)-3-methyl-1,2,3,4-tetrahydro-8-quinolinesulfonyl)-L-arginyl]-2-piperidinecarboxylic acid hydrate, a selective thrombin inhibitor, exerts a direct action on phenotype conversion of vascular smooth muscle cells, cultured rabbit aortic vascular smooth muscle cells were employed. Myosin heavy chain isoforms (SM1, SM2, and SMemb) mRNA expressions were evaluated by in situ hybridization and reverse transcription-polymerase chain reaction (RT-PCR). After the cells were incubated in serum-free medium containing argatroban (10 and 50 microg/ml) and platelet-derived growth factor (PDGF)-BB (10 and 50 ng/ml) for 3 h, total RNA was extracted. In situ hybridization demonstrated that myosin heavy-chain isoform mRNAs were homogenously expressed in argatroban- and PDGF-BB-treated cells. RT-PCR revealed that SM1/SM2 mRNA expressions were not changed with argatroban, while SMemb mRNA expression was increased to 1.6-fold with a statistical significance (P<0.05). Treatment with argatroban (10 and 50 microg/ml) at 24 h did not change SM1/SM2 mRNA expressions. Although SMemb mRNA expression was slightly increased, there was no statistical significance. Other phenotype markers including plasminogen activator inhibitor-1 (PAI-1) and beta-actin mRNAs were also significantly increased by argatroban. In conclusion, argatroban can directly induce phenotype conversion of vascular smooth muscle cells with the resultant up-regulation of SMemb, PAI-1, and beta-actin mRNAs.

Mast cell-derived VEGF enhances the passage of IgE FE-3 through the rat aortic endothelial cell monolayer.

BACKGROUND: It is well known that the IgE-mediated allergic reaction in various extravascular tissues is induced by the mutual interaction of IgE, target cells (mast cells, MCs) and allergens. However, so far little has been known about the detailed mechanism whereby IgE in the circulating blood is transferred to the extravascular tissue. To examine whether or not MCs are involved in the permeability of IgE across rat aortic endothelial cells (RAECs) in vitro, we determined the permeability constant (PC) of dinitrophenyl-specific rat IgE (IgE FE-3) using a dual chamber system. METHODS: Isolated RAECs obtained by a primary explant technique were seeded on a collagen-coated membrane in the upper chamber. MCs were collected from the peritoneal cavity of Wistar rats and suspended in the lower chamber. The time-dependent changes in concentration of IgE FE-3 (IgE) in the upper and lower chambers were measured by IgE capture ELISA after addition of IgE (10 microg/ml) to the upper chamber. RESULTS: The cultured medium of IgE-stimulated MCs significantly increased the PC of IgE (9.86 +/- 0.46 x 10(-6) cm/s), as compared to the value to which calcium ionophore A 23187-stimulated MCs increased the PC of IgE (7.97 +/- 0.21 x 10(-6) cm/s). The increase of PC by IgE-stimulated MCs was most strongly inhibited by suramin (92.3% +/- 1.89), and was weakly inhibited by tranexamic acid, cimetidine and diphenhydramine. In addition, the PC of IgE was increased with the increase in the MC-derived vascular endothelial growth factor/permeability factor (VEGF). CONCLUSIONS: After IgE is transferred from the circulating blood to the extravascular tissue, it may bind to Fc epsilon RI of the MC and the other Fc epsilon RI-bearing cells. The MC is then activated through the interaction of IgE and Fc epsilon RI. Release of VEGF from the activated MC increases, and the VEGF enhances the permeability of IgE.

An antibody that binds to primary specific pocket-associated structure in the active site of bovine thrombin.

We attempted to produce a monoclonal antibody (MAb) against the active site of native thrombin. Bovine thrombin was treated with diisopropyl fluorophosphate, and prepared diisopropylphosphoryl-thrombin was used for the immunization to BALB/c mice. Spleen cells of immunized mice were hybridized with mouse myeloma cells P3U1, and a hybridoma clone CC2, which produced a MAb against bovine thrombin was established. The MAb produced by hybridoma clone CC2 (MAb(CC2)), consisting of IgG(1) and kappa light chain, was purified using protein A affinity chromatography. Purified MAb(CC2) prolonged the fibrin forming time of bovine thrombin and inhibited the release of fibrinopeptide A from rabbit fibrinogen. In addition, it was found that argatroban partially, but competitively, interfere the binding between MAb(CC2) and bovine thrombin. It was then considered that MAb(CC2) would bind to the molecular structure associating primary specific pocket in the active site of bovine thrombin.

Azelastine and suplatast shorten the distribution half-life of IgE in rats.

We aim to clarify whether suplatast and azelastine (anti-allergic drugs) can shorten the half-life of imnunoglobulin E (IgE) in the circulating blood. Thirty Wistar rats were divided into six groups. Distilled water or anti-allergic drugs were given orally for 6 days after the first sensitization. Two milligrams of monoclonal dinitrophenyl (DNP)-specific rat IgE was administered to the rats, which had been given suplatast or azelastine orally. The level of DNP-specific rat IgE in the serum was estimated by IgE-capture enzyme-linked immunosorbent assay, and the turnover of IgE was analyzed from its pharmacokinetic parameters. The elimination half-life of rat IgE was about 12 h irrespective of the sensitized state. The intercompartmental rate constants (Kct and Ktc) in the suplatast-administered or azelastine-administered group were larger than those of the distilled water-administered group under non-sensitized conditions. These findings suggested that the anti-allergic drugs used in the present study facilitated the excretion of IgE from the circulation in rats.

Bound thrombin from crushed clots is composed of alpha-thrombin and the N-terminal regions of alpha- and gamma-chains of fibrinogen.

We aimed at clarifying the structural characteristics of the bound thrombin that is liberated by mechanical breakdown of fibrin clots. Fibrin clots were prepared with bovine thrombin and rabbit fibrinogen, and were crushed mechanically with a glass rod. The supernatant of the crushed clots was subjected to immunoaffinity chromatography to isolate the bound thrombin. Western blotting analysis revealed that the bound thrombin could be reacted with both antithrombin and antifibrinogen under unreduced conditions. SDS-PAGE under reduced conditions revealed that there were three bands, two of which were found to be the N-terminal fragments of the alpha- and gamma-chains of fibrinogen. The bound thrombin could be dissociated into three distinct fibrin fragments and bovine alpha-thrombin when denatured by 8 M urea. Thus, the bound thrombin liberated from crushed clots is a stable complex between bovine alpha-thrombin and fibrin fragments of the N-terminal regions of rabbit alpha- and gamma-chains.