Hiroyuki Matsuno

Simvastatin stimulates VEGF release via p44/p42 MAP kinase in vascular smooth muscle cells

It has been shown that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) modulate vascular smooth muscle cell functions. In the present study, we investigated the effect of simvastatin on vascular endothelial growth factor (VEGF) release, and the underlying mechanism, in a rat aortic smooth muscle cell line, A10 cells. Administration of simvastatin increased the VEGF level in rat plasma in vivo. In cultured cells, simvastatin significantly stimulated VEGF release in a dose-dependent manner. Simvastatin induced the phosphorylation of p44/p42 MAP kinase but not p38 MAP kinase or SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase). PD98059 and U-0126, inhibitors of the upstream kinase that activates p44/p42 MAP kinase, significantly reduced the simvastatin-induced VEGF release in a dose-dependent manner. The phosphorylation of p44/p42 MAP kinase induced by simvastatin was reduced by PD98059 or U-0126. Moreover, a bolus injection of PD98059 truly suppressed the simvastatin-increased VEGF level in rat plasma in vivo. These results strongly suggest that p44/p42 MAP kinase plays a role at least partly in the simvastatin-stimulated VEGF release in vascular smooth muscle cells.

Inhibitors of fibrinolytic components play different roles in the formation and removal of arterial thrombus in mice.

Plasminogen activator inhibitor-1 (PAI-1) and &agr;2-anti-plasmin (&agr;2-AP) may contribute to arterial thrombolysis resistance. The role of these components on thrombus evolution in vivo was investigated in mice deficient for PAI-1 (PAI-1 −/− ) or &agr;2-AP (&agr;2-AP −/− ) or their wild-type counterparts (PAI-1 +/+, &agr;2-AP +/+ ). Moreover, the influence of either PAI-1 or &agr;2-AP deficiency on the results of pharmacologic inhibition of glycoprotein IIb/IIIa of platelets or thrombin was also investigated. A thrombus was induced in the murine carotid artery by endothelial injury. The &agr;2-AP −/− mice were indistinguishable from wild-type, whereas the time to occlusion in PAI-1 −/− was significantly prolonged to 24.9 ± 3.7 min. Vascular patency was markedly increased in both PAI-1- and &agr;2-AP-deficient mice. In separate animals, either a glycoprotein IIb/IIIa antagonist or a thrombin inhibitor was applied. The time required to occlusion was prolonged in a dose-dependent manner in all types of mice. When each compound was administered to PAI-1 −/− mice, significant changes were observed. In conclusion, lack of PAI-1 prolongs the time to occlusion and accelerates clot lysis, whereas &agr;2-AP only has an effect on spontaneous reperfusion. Consequently, the inhibition of PAI-1, but not of &agr;2-AP, could enhance the effects of anti-thrombotic therapy.

α2‐Antiplasmin plays a significant role in acute pulmonary embolism

Summary.u2002 The importance of pulmonary embolism (PE) due to venous thrombosis is recognized in the treatment of vascular diseases. We have investigated the physiological effects of plasmin generation in experimental acute PE using mice deficient in plasminogen (Plg−/−) or α2‐antiplasmin (α2‐AP−/−). PE was induced by continuous induction of venous thrombus in the left jugular vein by endothelial injury due to photochemical reaction. The mortality of wild‐type mice was 68.8% at 2u2003h after the initiation of venous thrombosis and it was significantly reduced in α2‐AP−/− mice (41.7%). In contrast, Plg−/− mice did not survive. Histological evidence of thromboembolism in the lung was obtained in all mice. However, whereas a strict thromboembolism was observed in Plg−/− mice, only a few thrombi were detected in the lungs of α2‐AP−/− mice. Plasma fibrinogen levels measured in mice were not different. When α2‐AP was infused in α2‐AP−/− mice, the mortality was indistinguishable from wild‐type mice. Tissue‐type plasminogen activator (tPA) did not reduce the mortality due to acute PE in wild‐type mice. However, in α2‐AP−/− mice, tPA (0.52u2003mgu2003kg−1) significantly decreased the mortality compared with that of α2‐AP−/− mice without tPA. The bleeding time was not significantly prolonged in either type of mice treated with tPA. The lack of plasminogen increases the mortality due to acute PE while a lack of α2‐AP decreases the mortality rate, which can be further reduced by tPA administration. Therefore, the combination of inhibition of α2‐AP with thrombolytic therapy could be beneficial in the treatment of acute PE.

Lack of α2-Antiplasmin Enhances ADP Induced Platelet Micro-Aggregation through the Presence of Excess Active Plasmin in Mice

AbstractBackground: The role of α2-antiplasmin (α2-AP) on platelet aggregation was investigated using mice deficient in α2-AP (α2-AP−/−) or using wild type mice (α2-AP+/+).nMethods: Blood samples were taken from each mouse under anesthesia with ether and platelet rich plasma (PRP) was prepared. Platelet aggregation induced by various doses of ADP (0.3–30 μM) was detected using a laser-light scattering (LS) system. Aggregated forms were observed using a scanning electron microscopy (SEM).nResults: Dose-dependent platelet aggregation was not different in both types of mice. However, platelet micro-aggregate formation in α2-AP−/− mice induced by low dose of ADP (1.0 μM) markedly increased compared to the situation in wild type mice. Aggregated form detected by SEM showed supported data from LS analysis. When washed platelets of α2-AP+/+ mice were resuspended in plasma of α2-AP−/− mice, platelet micro-aggregation was also increased. On the contrary, when washed platelets of α2-AP−/− mice were suspended in plasma of α2-AP+/+ mice, platelet micro-aggregation did not change. In separate experiments, tPA (1.0 μg/ml) was added to PRP before the stimulation of ADP. tPA had no effect on platelet aggregation in α2-AP+/+ mice, however platelet micro-aggregation in α2-AP−/− mice was markedly increased by the treatment with tPA. Moreover, the amount of released ATP from stimulated platelets was increased in α2-AP−/− mice treated with tPA.nConclusion: Lack of α2-AP increased platelet micro-aggregation, and plasmin plays an important role in the formation of platelet aggregation when α2-AP knockout mice are used. Consequently, the reduction of α2-AP could be a risk factor for the activation of platelets resulting in thrombus formation.

Platelet‐derived growth factor‐BB phosphorylates heat shock protein 27 in cardiac myocytes

It is recognized that heat shock protein 27 (HSP27) is highly expressed in heart. In the present study, we investigated whether platelet‐derived growth factor (PDGF) phosphorylates HSP27 in mouse myocytes, and the mechanism underlying the HSP27 phosphorylation. Administration of PDGF‐BB induced the phosphorylation of HSP27 at Ser‐15 and ‐85 in mouse cardiac muscle in vivo. In primary cultured myocytes, PDGF‐BB time dependently phosphorylated HSP27 at Ser‐15 and ‐85. PDGF‐BB stimulated the phosphorylation of p44/p42 mitogen‐activated protein (MAP) kinase, p38 MAP kinase, and stress‐activated protein kinase/c‐Jun N‐terminal kinase (SAPK/JNK) among the MAP kinase superfamily. SB203580, a specific inhibitor of p38 MAP kinase, reduced the PDGF‐BB‐stimulated phosphorylation of HSP27 at both Ser‐15 and ‐85, and phosphorylation of p38 MAP kinase. However, PD98059, a specific inhibitor of MEK, or SP600125, a specific inhibitor of SAPK/JNK, failed to affect the HSP27 phosphorylation. These results strongly suggest that PDGF‐BB phosphorylates HSP27 at Ser‐15 and ‐85 via p38 MAP kinase in cardiac myocytes.

Pharmacokinetic and Pharmacodynamic Properties of a New Thromboxane Receptor Antagonist (Z‐335) after Single and Multiple Oral Administrations to Healthy Volunteers

The pharmacokinetics and pharmacodynamics of a new oral thromboxane (TX) A2 receptor antagonist, Z‐335, were studied in healthy male volunteers following single doses (0.5–40 mg, PO) in a dose‐escalating manner and multiple doses (40 mg, PO, once daily for 7 consecutive days) with a single‐blind, placebo‐controlled design. Serial blood and urine samples were analyzed for Z‐335 and its metabolites to obtain key pharmacokinetic parameters. In the single‐dose (10, 20, and 40 mg) study, the maximum plasma concentration (Cmax) and area under the plasma concentration versus time curve (AUC) increased in proportion to the dose when administered after fasting, while the mean elimination half‐life (t1/2β) was essentially unchanged (7.79‐7.93 h). Recovery of the unchanged and taurine‐conjugated drugs in the urine within 24 hours was 6.5% to 8.4% and 11.9% to 14.2%, respectively. These parameters essentially remained unchanged when the effect of meal intake was evaluated at the dose of 20 mg with a crossover design. Ex vivo platelet aggregation in the plasma by a TXA2 analogue, U46619, was completely inhibited within 2 hours after all doses, and complete inhibition was maintained for 12 to 14 hours, depending on the dose. The aggregation induced by collagen was also inhibited to a lesser extent, whereas that by adenosine diphosphate was hardly influenced. In themultiple‐dose study, Cmax and AUC0–24 were increased by 34% after the last dose compared with the first dose. Z‐335 afforded extensive inhibition of platelet aggregation by U46619 throughout the administration period, which returned, however, almost to the control level 48 hours after the last dose. The agent was well tolerated without any abnormalities in subjective and objective symptoms, blood biochemistry, hematology, and urinalysis definitely attributable to the agent, except for the changes expected from its TXA2 receptor‐antagonizing actions. Z‐335 was concluded to be safe and to provide long‐lasting blockade of TXA2 receptors on the basis of a once‐daily regimen, promoting further clinical evaluation.

Midazolam Stimulates Vascular Endothelial Growth Factor Release in Aortic Smooth Muscle Cells Role of the Mitogen-activated Protein Kinase Superfamily

Background Intravenous anesthetics used during perioperative periods affect the vascular signaling molecules and the vascular reactivity. Vascular endothelial growth factor (VEGF), an angiogenesis factor produced in and secreted from aortic smooth muscle cells, is a specific mitogen for vascular endothelial cells. This study investigated the effects of various intravenous anesthetics on VEGF release, and the underlying mechanism, in a rat aortic smooth muscle cell line, A10 cells. Methods Intravenous anesthetics (midazolam and propofol) were continuously administered to rats by infusion. Cultured A10 cells were stimulated by intravenous anesthetics (midazolam, propofol, and ketamine). VEGF was evaluated by immunoassay. The phosphorylation of mitogen-activated protein (MAP) kinases was evaluated by Western blotting. Results Continuous infusion of midazolam, but not propofol, increased the VEGF concentration in rat plasma. In cultured cells, midazolam stimulated VEGF release, but propofol and ketamine did not. Midazolam induced phosphorylation of p44/p42 MAP kinase and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK), without affecting p38 MAP kinase. PD98059 and U0126, specific inhibitors of MAP kinase kinase, significantly reduced the midazolam-stimulated release of VEGF. SP600125, a specific inhibitor of SAPK/JNK, significantly reduced midazolam-stimulated VEGF release. Applied together, PD98059 and SP600125 produced an additive reduction in midazolam-stimulated VEGF release. Moreover, a bolus injection of PD98059 truly inhibited the midazolam-increased VEGF concentration in rat plasma in vivo. Conclusions Midazolam, but not propofol or ketamine, stimulates VEGF release in aortic smooth muscle cells. Its effect is mediated at least in part via activation of p44/p42 MAP kinase and SAPK/JNK.

Characterization of simple and reproducible vascular stenosis model in hypercholesterolemic hamsters

The importance of low-density lipoprotein (LDL) in the etiology of atherosclerosis is well recognized. We have established a reproducible stenosis model in hypercholesterolemic hamsters, and the process of arterial stenosis by thrombus or neointima was studied and compared with that in normal hamsters. The level of plasma LDL was 4.6 times higher in hamsters fed a high-cholesterol diet than in hamsters fed normal food. Endothelial injury in right common carotid arteries was induced using a modified catheter. Arterial blood flow was monitored continuously using a Doppler flow probe. Arterial patency after the initiation of injury in high-cholesterol hamsters was significantly changed as compared with that of normal hamsters. Neointima was observed 2 wk after the vascular injury. The neointimal area of high-cholesterol hamsters was significantly larger than that of normal hamsters. To characterize the stenosis in hypercholesterolemic hamsters, we measured platelet aggregation, thrombin time, activated partial thromboplastin time, and proliferating smooth muscle cells (SMC) in vitro and in vivo. The half-maximal inhibitory concentration value for platelet aggregation induced by thrombin or collagen, the DNA synthesis stimulated by plateletderived growth factor (PDGF)-BB, and 5-bromo-2-deoxy-uridine labeling indices (proliferating index of SMC in vivo) in high-cholesterol hamsters were each significantly higher than the comparable value from normal hamsters. However, specific binding of PDGF-BB in SMC was not different between the two types of hamsters. Furthermore, we investigated the inhibitory effects of probucol or losartan on neointima formation using this model. Probucol, but not losartan, significantly reduced the neointimal area in hypercholesterolemic hamsters. These findings indicated that high levels of plasma LDL strongly contributed to the development of thrombus and neointima formation via both up-regulation of platelet aggregation and the enhancement of SMC proliferation. This stenosis model may be useful for the investigation of hypercholesterolemia-associated cardiovascular diseases.