Biomedical subjects

Takahisa Masaki

Publications and source records attributed to Takahisa Masaki.

15 recordsLinked to original sources

PubMed2007

Polyreactive myelin oligodendrocyte glycoprotein antibodies: Implications for systemic autoimmunity in progressive experimental autoimmune encephalomyelitis.

Two myelin oligodendrocyte glycoprotein (MOG92-106) monoclonal antibodies (mAbs) were produced from an A.SW mouse with progressive experimental autoimmune encephalomyelitis. Polyreactivity/specificity of the mAbs was demonstrated by ELISA. Functionality and a potential role in pathogenesis of systemic autoimmunity were demonstrated in vitro in a lymphocytotoxicity assay and in vivo upon injection into naïve mice. Injection of MOG mAb producing hybridomas into naïve mice resulted in immunoglobulin deposition in kidneys and liver. This model will be useful in determining whether transitional forms between CNS (organ)-specific and systemic autoimmune diseases exist, and whether progressive multiple sclerosis has features of a systemic autoimmune disease.

Animals↗

PubMed2006

Differential effects of imatinib on PDGF-induced proliferation and PDGF receptor signaling in human arterial and venous smooth muscle cells.

Platelet-derived growth factor (PDGF) has been implicated in smooth muscle cell (SMC) proliferation, a key event in the development of myointimal hyperplasia in vascular grafts. Recent evidence suggests that the PDGF receptor (PDGFR) tyrosine kinase inhibitor, imatinib, can prevent arterial proliferative diseases. Because hyperplasia is far more common at the venous anastomosis than the arterial anastomosis in vascular grafts, we investigated whether imatinib also inhibited venous SMC (VSMC) proliferation, and examined possible differences in its mechanism of action between VSMC and arterial SMC (ASMC). Human ASMC and VSMC were stimulated with PDGF-AB, in the presence or absence of imatinib (0.1-10 microM). Proliferation was assayed using the 5-bromo-2'-deoxyuridine (BrdU) incorporation assay, while PDGFR, Akt and ERK1/2-mitogen activated protein kinase (MAPK) signaling pathways were investigated by immunoblotting. The proliferative response to PDGF at 50 and 100 ng/ml was 32 and 43% greater, respectively, in VSMC than in ASMC. Similarly, PDGF-stimulated proliferation was more sensitive to inhibition by imatinib in VSMC than ASMC (IC(50) = 0.05 microM vs. 0.4 microM; P < 0.01). Imatinib also more effectively inhibited PDGF-induced phosphorylation of PDGFRbeta and Akt in VSMC, compared to ASMC. These data highlight inherent pharmacodynamic differences between VSMC and ASMC in receptor and cell signaling functions and suggest that imatinib therapy may be useful for the prevention of venous stenosis in vascular grafts.

Arteries↗

PubMed2006

Different signaling responses to anti-proliferative agents in human aortic and venous smooth muscle cells.

Proliferation of smooth muscle cells (SMCs) contributes to the stenosis of coronary arteries and vascular grafts. Local delivery of anti-proliferative drugs can prevent vascular stenosis. To understand the cellular responses to anti-proliferative agents, we investigated the signaling events in cultured human aortic SMCs (ASMCs), saphenous venous SMCs (VSMCs), and dermal fibroblasts (DFs) in response to paclitaxel or etoposide. Cellular mitochondrial and proliferative activities were examined with the methylthiazoletetrazolium (MTT) dye reduction and the bromodeoxyuridine (BrdU) incorporation assay, respectively. Cell proliferation was almost completely suppressed by paclitaxel or etoposide, but apoptosis was achieved in only about 50% of cells at the highest drug concentrations, suggesting the presence of compensatory mechanisms to prevent apoptosis. Examination of three important signaling pathways revealed significant differences between ASMCs, VSMCs, and DFs. Treatment with either paclitaxel or etoposide caused a transient phosphorylation/activation of p42 MAPK in ASMCs and DFs, but had no effect on phospho-p42/44 MAPK in VSMCs. High-dose etoposide enhanced p38 MAPK activation in ASMCs, but not in VSMCs. The p38 inhibitor, PD169316, partially inhibited etoposide-induced ASMC apoptosis, but induced apoptosis in VSMCs. The effects of etoposide and paclitaxel on Akt also differed between ASMCs and VSMCs. These observations indicate that ASMCs and VSMCs differ in the response of signaling pathways to anti-proliferative agents. In ASMCs, p42/44 MAPK appears to serve a pro-survival role, whereas p38 MAPK is a pro-apoptotic regulator. In contrast, p38 MAPK is an important pro-survival regulator in VSMCs and p42/44 MAPK appears to play a minor role in responding to anti-proliferative drugs.

Animals↗

PubMed2006

Cellular pharmacokinetics and pharmacodynamics of dipyridamole in vascular smooth muscle cells.

Hemodialysis arteriovenous grafts are often plagued by stenosis at the vein-graft anastomosis, which is due to the proliferation of venous smooth muscle cells (SMCs). Perivascular delivery of dipyridamole, a potent antiproliferative agent, has been proposed for the prevention of graft stenosis. In order to develop an optimal delivery system for dipyridamole, we examined its pharmacokinetics and pharmacodynamics in human and porcine venous and arterial SMCs in vitro. SMCs were incubated with dipyridamole for various durations, and visualized for the uptake and release by fluorescence microscopy, which were further quantified by fluorospectrometry. The antiproliferative effect of dipyridamole was examined by cell counting or the methylthiazoletetrazolium (MTT) dye-reduction assay. Cytotoxicity was examined by the lactate dehydrogenase (LDH)-release assay. The kinetics of dipyridamole transport through the cell membrane was compatible with a passive diffusion mechanism. Dipyridamole inhibited SMC proliferation in a dose-dependent manner and was more effective in venous than arterial cells in both species. The inhibition was completely reversible at 15microg/ml upon drug removal from the medium. At 25microg/ml, however, the effect was partially irreversible, which might be attributed to the cytotoxicity of dipyridamole. These data support the need for sustained delivery of dipyridamole to achieve the long-term inhibition of SMC proliferation in the prevention of stenosis since SMCs are continuously stimulated at the anastomosis of hemodialysis arteriovenous grafts.

Animals↗

PubMed2006

Taste aversion in rats induced by forced swimming, voluntary running, forced running, and lithium chloride injection treatments.

The present experiment compared the strengths of taste aversion learning in rats induced by forced swimming in a water pool (5, 15, 30, or 60 min), voluntary running in an activity wheel (15, 30, 60, or 120 min), forced running in a motorized wheel (60 min at the speed of 8 m/min), optional running in the apparatus consisting of an activity wheel and a side room (120 min), and a lithium chloride (LiCl, 0.15 M LiCl at 2% of body weight) injection. The rats were given an access to saccharin solution immediately followed by one of the above treatments or simply returned back to the home cages for the control group. On the next 2 days, aversion to the saccharin solution was assessed by two-bottle choice testing between it and tap water. The following results were obtained. (1) The saccharin aversion was a positive function of exercise durations in the forced swimming and voluntary running rats, and the exercise of more than 30 min induced statistically significant saccharin aversion, compared with the control rats. (2) The forced running caused relatively strong saccharin aversion. The group of forced running rats acquired the numerically strongest saccharin aversion on average among all exercised rats. (3) The optional running treatment had little effect. (4) The LiCl injection resulted in the strongest aversion among the all treatments explored here.

Animals↗

PubMed2006

Perivascular tissue pharmacokinetics of dipyridamole.

PURPOSE: The tissue diffusivity (D(g)) and partitioning (K) for dipyridamole were determined and a model was developed to examine the relationship between perivascular dose and local dipyridamole tissue concentrations. METHODS: Experiments were performed using an in vitro perfusion apparatus that recirculated buffer through different graft samples or normal porcine femoral arteries and veins. The grafts or blood vessels were immersed in a compartment containing Krebs-Henseleit (KH) buffer and dipyridamole (30 microg/mL). The recirculating buffer was sampled at multiple time points and dipyridamole was assayed. Estimates of the effective diffusivity (D(g)) and partition coefficient (K) of the drug in the vessel wall were determined and used to simulate dipyridamole tissue concentration after perivascular delivery. RESULTS: Dipyridamole diffusivity within native femoral veins (D(g) = 3.87 +/- 0.93 x 10(-6) cm2/s) was approximately twice that within femoral arteries (D(g) = 2.06 +/- 0.79 x 10(-6) cm2/s, p < 0.01). Explanted grafts showed the lowest diffusivity. Partition coefficients of femoral arteries (K = 4.11 +/- 0.99) were higher than those of femoral veins (K = 2.05 +/- 0.85, p < 0.01) and explanted graft (K = 0.89 +/- 0.56, p < 0.01). DISCUSSION: The results demonstrate that local drug kinetics vary greatly for different types of blood vessels and grafts. The pharmacokinetic parameters and resulting computational simulations are helpful in exploring perivascular drug delivery strategies.

Algorithms↗

PubMed2006

Development of a sustained-release system for perivascular delivery of dipyridamole.

Vascular access grafts implanted in dialysis patients are prone to failure in the long-term because of stenosis and occlusion caused by neointimal hyperplasia. Local delivery of antiproliferative drugs may be effective to prevent this consequence while minimizing the systemic side effects they cause. We developed a combination of poly(lactide-co-glycolide) (PLGA) microspheres with ReGel, an injectable copolymer, as a sustained-release system for perivascular delivery of an antiproliferative drug, dipyridamole. Dipyridamole-incorporated PLGA microspheres with various molecular weights (MWs) of PLGA were prepared by oil-in-water emulsion method. Encapsulation efficiency and surface morphology of microspheres were characterized. In vitro release kinetics of dipyridamole from ReGel or from microspheres/ReGel was experimentally determined. Without microspheres, 40% of the dipyridamole was released from ReGel as an initial burst in the first 3 days followed by continuous release in the subsequent 2 weeks. The use of PLGA microspheres decreased the initial burst and extended dipyridamole release from 23 to 35 days with increasing MW of PLGA. The highest MW PLGA showed a lag time of 17 days before consistent drug release occurred. Mixing microspheres and ReGel with two different MW PLGA achieved a continuous release for 35 days with little initial burst. In vivo release of dipyridamole from microspheres/ReGel exhibited a comparable release pattern to that seen in vitro. This injectable platform is a promising technique for sustained perivascular delivery of antiproliferative drugs.

Animals↗

PubMed2006

Mechanism of dipyridamole's action in inhibition of venous and arterial smooth muscle cell proliferation.

Dipyridamole is a potential pharmacological agent to prevent vascular stenosis because of its antiproliferative properties. The mechanisms by which dipyridamole inhibits the growth of vascular smooth muscle cells, especially venous smooth muscle cells, are unclear. In the present study, dipyridamole transiently but significantly increased cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) levels in human venous and arterial smooth muscle cells in a time- and dose-dependent manner. Peak concentrations of both cyclic nucleotides were achieved at 15-30 min. and correlated with inhibition of proliferation in both cell types. The antiproliferative effects of dipyridamole observed at 48 hr were similar whether drug exposure was only 15 min. or sustained for 48 hr. Specific competitive inhibitors of protein kinases A and G attenuated the antiproliferative effects of subsaturating concentrations of dipyridamole, with the effects of protein kinase inhibition being particularly pronounced in venous smooth muscle cells. Flow cytometry analysis showed that dipyridamole caused an enrichment of cells in G(0)/G(1) and a corresponding reduction of cells in S phase. These data indicate that a transient increase in cGMP and cAMP is sufficient to induce downstream kinase activation and subsequent cell cycle arrest, and that protein kinase G may be more important than protein kinase A in mediating the growth inhibitory effect of dipyridamole in venous protein kinase.

Aorta↗

PubMed2005

Different responses by cultured aortic and venous smooth muscle cells to gamma radiation.

BACKGROUND: Stenosis of hemodialysis arteriovenous grafts is usually focal and caused by the proliferation of vascular smooth muscle cells (SMCs). External radiation of the graft is a potential strategy to prevent stenosis; however, the relative responsiveness of arterial and venous SMCs to radiation is unknown. METHODS: Human aortic and saphenous vein SMCs were cultured in a medium containing growth factors and serum and treated with 0 to 50 Gy in a gamma irradiator. At 2 to 20 days post-irradiation, cell counting, methylthiazoletetrazolium dye reduction, [(3)H]-thymidine uptake, and bromodeoxyuridine (BrdU) incorporation assays were performed. RESULTS: All assays showed that 1 to 50 Gy inhibited the proliferation of both aortic and venous SMCs in a dose-dependent manner. Importantly, venous cells were less susceptible to radiation in all assays, compared to aortic cells. At day 10, 1 to 50 Gy of radiation inhibited the increase in the number of aortic cells by 24% to 66% and venous cells by 8% to 25% (P < 0.01) (aortic vs. venous). The differences between aortic and venous cells varied among different assays and were most pronounced in the BrdU assay. CONCLUSION: Inasmuch as myointimal hyperplasia occurs at both arterial and venous anastomoses, future strategies using radiation to prevent hemodialysis vascular access stenosis should take these differences into consideration.

Antimetabolites↗

PubMed2004

Taste aversion learning induced by delayed swimming activity.

The experiment reported here demonstrated that forced swimming endows rats with aversion to a taste solution consumed 30 min before the swimming. The experimental rats were allowed to drink 0.2% sodium saccharin solution, which was followed by a 30-min empty interval, and then a 20-min swimming opportunity in water. Compared with the control rats, which were returned to their home cages after drinking the saccharin, the experimental rats drank a small amount of saccharin solution both in the later sessions of one-bottle training and in the subsequent two-bottle choice (saccharin versus tap water) testing. The delayed swimming procedure was as effective as an immediate swimming procedure, extending the generality of the swimming-induced taste aversion, which we recently discovered with the immediate swimming procedure.

Animals↗

PubMed2004

Further evidence for conditioned taste aversion induced by forced swimming.

A series of experiments with rats reported that aversion to a taste solution can be established by forced swimming in a water pool. Experiment 1 demonstrated that correlation of taste and swimming is a critical factor for this phenomenon, indicating associative (i.e., Pavlovian) nature of this learning. Experiment 2 showed that this learning obeys the Pavlovian law of strength, by displaying a positive relationship between the duration of water immersion in training and the taste aversion observed in subsequent testing. Experiment 3 revealed that swimming rather than being wet is the critical agent, because a water shower did not endow rats with taste aversion. Experiment 4 found that taste aversion was a positive function of water level of the pools in training (0, 12 or 32 cm). These results, taken together, suggest that energy expenditure caused by physical exercise might be involved in the development of taste aversion.

Analysis of Variance↗

PubMed2004

Arterial and venous smooth-muscle cells differ in their responses to antiproliferative drugs.

Arteriovenous polytetrafluoroethylene (PTFE) grafts used for hemodialysis often fail as the result of myointimal hyperplasia with vascular smooth-muscle-cell (SMC) proliferation. The stenotic lesions occur primarily at the graft-vein anastomosis and less frequently at the graft-artery anastomosis. To explore the potentials of pharmacologic agents in preventing hemodialysis-graft stenosis, we first examined the susceptibility of venous and aortic SMCs to 3 antiproliferative drugs. Human aortic and saphenous-vein SMCs were cultured in a medium containing insulin, epidermal growth factor, fibroblast growth factor, and fetal bovine serum. Various concentrations of dipyridamole (0-100 microg/mL), paclitaxel (0-100 microg/mL), and tranilast (0-300 microg/mL) were added. After 72 hours, we subjected the cells to a mitochondrial enzymatic (methylthiazoletetrazolium; MTT) assay and a bromodeoxyuridine (BrdU)-incorporation assay as a means of assessing their proliferation. Dipyridamole, paclitaxel, and tranilast each inhibited the proliferation of aortic and venous SMCs in a dose-dependent manner ( P <.0001). Approximately 90% inhibition was achieved at dipyridamole concentrations of 75 microg/mL and greater in both MTT and BrdU assays; paclitaxel and tranilast were less effective. The venous SMCs were substantially more susceptible to inhibition by all 3 drugs than were the aortic SMCs in the MTT assay. The concentrations required to produce 50% inhibition (IC 50 ) in the venous cells were 5.8 microg/mL (11.5 micromol/L), 9.1 microg/mL (10.7 micromol/L), and 37.4 microg/mL (114.3 micromol/L), respectively, for dipyridamole, paclitaxel, and tranilast. These concentrations were approximately 4.2, 5.3, and 3.0 times lower, respectively, than the corresponding IC 50 values for the aortic cells. The differences in IC 50 between the aortic and venous cells for the 3 drugs were less pronounced in the BrdU assay. The results of this study suggest that strategies for the prevention of stenosis should take into account the fact that lesions at venous anastomoses of arteriovenous grafts may respond differently to drugs than do those at arterial anastomoses.

Arteries↗

PubMed2004

Taste aversion learning induced by forced swimming in rats.

Two experiments demonstrated that forced swimming endowed rats with aversion to the taste solution consumed before the swimming. In Experiment 1, the rats given a trial of taste-swimming sequence drank less of the taste solution in a later test than did the rats given a taste-alone trial. The rats given a trial of taste-poisoning-swimming sequence, however, drank more of the taste solution in the testing than did the rats given a trial of taste-poisoning sequence. These results suggest that some effects of swimming (e.g., energy expenditure caused by physical exercise) induce conditioned taste aversion although they attenuate taste aversion conditioned by poisoning. The attenuation of poison-induced taste aversion by swimming has been reported in the literature, but the swimming-induced taste aversion is novel. Experiment 2, accordingly, was planned to confirm this phenomenon with a differential conditioning procedure, where one of two taste solutions was paired with swimming while the other was not. After a few repetitions of these two types of trials, the rats' intakes of these two solutions were differentiated to show that swimming has the ability to cause taste aversion.

Animals↗

PubMed2004

Inhibition of neointimal hyperplasia in vascular grafts by sustained perivascular delivery of paclitaxel.

BACKGROUND: Neointimal hyperplasia occurs commonly at the anastomoses of arteriovenous grafts for chronic hemodialysis, causing stenosis and occlusion. Antiproliferative drugs may be effective in inhibiting hyperplasia, but local drug delivery would be required to minimize systemic side effects. We examined the feasibility of local drug delivery to inhibit neointimal hyperplasia at dialysis grafts in a canine model. METHODS: Bilateral polytetrafluoroethylene loop grafts (10-cm length and 6-mm internal diameter) were placed between the femoral artery and ipsilateral femoral vein of five mongrel dogs. At the time of surgery or 1 to 5 weeks later, 2 mL of a thermosensitive biodegradable copolymer (ReGel) mixed with 0.26 mg or 0.65 mg paclitaxel were applied to the external surface of one graft around the anastomoses to provide a depot for sustained release of the drug. ReGel alone without paclitaxel was applied to the contralateral graft as a control. The grafts and the connecting vessels were explanted at eight or nine weeks, and the cross-sections were examined histologically. The degree of hyperplasia at the anastomoses was graded by five blinded independent reviewers, with scores ranging from 0 to 5. RESULTS: The median (25th-75th percentile) hyperplasia score of both arterial and venous anastomoses was 1.80 (0.90-3.05) in the grafts treated with ReGel alone, and 0.95 (0.70-1.50) in the grafts treated with ReGel/paclitaxel (N= 8; P < 0.05 by Wilcoxon signed rank test). There were no noticeable localized or systemic complications attributed to the treatments in these animals. Paclitaxel levels in the plasma obtained from forelimb veins were undetectable (<10 ng/mL). CONCLUSION: These results suggest that the local delivery of antiproliferative agents using a thermosensitive, injectable biodegradable copolymer (ReGel) for sustained delivery is a promising strategy to inhibit neointimal hyperplasia of arteriovenous hemodialysis grafts.

Animals↗

PubMed2004

In vitro pharmacological inhibition of human vascular smooth muscle cell proliferation for the prevention of hemodialysis vascular access stenosis.

BACKGROUND: Vascular access for chronic hemodialysis often fails as a result of stenosis caused primarily by the proliferation of vascular smooth muscle cells (VSMC). Various drugs have been shown to inhibit the proliferation of VSMC under different conditions. METHODS: In this study, we compared the inhibitory effect of ten drugs on the proliferation of human aortic smooth muscle cells (SMC) in culture. Quiescent cells were cultured in the presence of growth factors, fetal bovine serum and incremental concentrations of the test drug. Cell proliferation was assessed by the MTT reduction assay. RESULTS: Aspirin, enalaprilat, heparin, hydroxyurea, indomethacin and tirofiban were ineffective. While dipyridamole, paclitaxel, tranilast and verapamil inhibited cell proliferation, the concentrations required were significantly higher than the clinical plasma levels achieved after systemic administration. CONCLUSION: Local delivery of these drugs to the target site may therefore be a more effective and appropriate strategy for the prevention of hemodialysis vascular access stenosis.

Antineoplastic Agents↗