Keita Shibata

Distinct Effects of Tissue-Type Plasminogen Activator and SMTP-7 on Cerebrovascular Inflammation Following Thrombolytic Reperfusion

Background and Purpose— Thrombolysis therapy using tissue-type plasminogen activator (t-PA) is occasionally accompanied by harmful outcomes, including intracerebral hemorrhage. We have reported that Stachybotrys microspora triprenyl phenol-7 (SMTP-7), a candidate thrombolytic drug, has excellent therapeutic effect on cerebral infarction in embolic stroke model in mice; however, little is known regarding whether this agent influences cerebrovascular inflammation following thrombolytic reperfusion. The current study aimed to compare the effects of recombinant t-PA (rt-PA) and SMTP-7 on cerebrovascular inflammation. Methods— The impact of rt-PA- and SMTP-7-induced thrombolytic reperfusion on leukocyte dynamics was investigated in a photochemically induced thrombotic middle cerebral artery occlusion (tMCAo) model in mice. Results— Both rt-PA and SMTP-7 administration in tMCAo mice (each 10 mg/kg) resulted in thrombolytic reperfusion. The SMTP-7-administered mice showed relatively mild rolling and attachment of leukocytes to the vascular wall in the middle cerebral vein, with weak peroxynitrite reactions and proinflammatory gene expression (IL-1&bgr;, TNF-&agr;, ICAM-1, and VCAM-1); thus, a small infarct volume compared with rt-PA-administered mice. In vitro study suggested that rt-PA at 20 &mgr;g/mL, but not SMTP-7 at a similar concentration, promotes cytokine-induced reactive oxygen species generation in cultured endothelial cells; moreover, SMTP-7 suppressed cytokine-induced VCAM-1 induction in the cells and leukocyte/ endothelial cell adhesions. Conclusions— Relatively mild cerebrovascular inflammation and cerebral infarction in the SMTP-7 mice, compared with in rt-PA mice, is thought to be caused at least in part by direct antioxidative actions of SMTP-7 in ECs.

Shear stress-dependent effects of lysophosphatidic acid on agonist-induced vasomotor responses in rat mesenteric artery.

We have previously shown that lysophosphatidic acid (LPA), a bioactive plasma lysophospholipid, markedly accelerates shear stress-induced Ca2+ responses in cultured vascular endothelial cells (ECs). This study aimed to demonstrate the impact of LPA and luminal shear stress on vasomotor regulation in the isolated rat mesenteric artery (MA) using a videomicroscopic technique. Although the addition of LPA to the perfusate in a concentration range of 0.03-0.3 μM had no significant effect on the basal MA tone, LPA in a similar concentration range led to increased phenylephrine-induced MA contraction and reduced acetylcholine-induced MA relaxation under physiological shear conditions. These vasomodulatory actions of LPA, which vanished upon removal of ECs, were positively dependent on luminal shear stress levels and were markedly inhibited by the LPA receptor antagonist Ki16425, the cyclooxygenase inhibitor indomethacin, and the thromboxane A2 receptor antagonist SQ29548. These data thus suggest that LPA can modify the agonist-induced vasomotor responses in MAs in a shear stress-dependent manner. This effect of LPA was mediated through ECs, the LPA receptor, and cyclooxygenase/thromboxane A2 signaling.

Evaluation of the effects of a new series of SMTPs in the acetic acid-induced embolic cerebral infarct mouse model

Abstract We reported previously that Stachybotrys microspora triprenyl phenol‐7 (SMTP‐7) showed potential thrombolytic, anti‐inflammatory and anti‐oxidant effects that account for its excellent pharmacological activity such as having a wider therapeutic time window than tissue plasminogen activator (t‐PA) and a significant protection against hemorrhage. The aim of the present study was to evaluate and compare the effect of a new series of SMTPs in the acetic acid‐induced embolic cerebral infarct mouse model. Thrombotic occlusion was produced in mice by inducing the transfer of acetic acid‐induced thrombi from the right common carotid artery into the brain. SMTPs were evaluated by their effect on reducing infarct area, neurological score and edema. Furthermore, plasmin formation, anti‐inflammatory and anti‐oxidant activities were assessed by fibrin zymography, measuring pro‐inflammatory gene expression, and thiobarbituric acid reactive substances (TBARS) assay, respectively. Treatment with either SMTP‐22 or SMTP‐43 (10 mg/kg), which have similar plasmin formation, anti‐inflammatory and anti‐oxidant activities to SMTP‐7, resulted in reduced infarct area, neurological score and edema. Coexistence of all these three activities appears to be important for the treatment of embolic infarction because SMTP‐6, SMTP‐25, and SMTP‐44D (10 mg/kg), which are each missing at least one of the three functions, were not as effective. Therefore, these results indicate that SMTP‐22 and SMTP‐43 have potential as medicinal compounds for the treatment of embolic cerebral infarction.

Knowledge of pharmacy students about doping, and the need for doping education: a questionnaire survey

BackgroundAnti-doping activities are carried out on a global scale. Based on these activities, the specialty of “sports pharmacist,” which entails a deeper comprehension of doping, use of supplements, and appropriate drug use for athletes, was established in 2009 in Japan. It is difficult to say whether the education on doping is adequate for pharmacy students who will be eligible to become sports pharmacists. It is also unclear how well these students understand doping. Therefore, the aim of this study was to investigate pharmacy students’ current knowledge of appropriate drug use, doping and use of supplements, and to explore the need for further education on these topics.MethodsA questionnaire survey was conducted from July 3rd to August 2nd in 2014 at Showa University in Japan. A total of 406 respondents (2nd- to 6th-year students) were assessed as eligible. Group comparison was used to compare those who had attended a lecture about doping and those who had not.ResultsMost of the students only knew the word doping and had not attended a lecture on the subject, but 72% of them expressed a desire to attend one. Over half did not know that the most common doping violation in Japan is unintentional doping, and were unfamiliar with certain past cases of doping. In addition, 41% did not know that over-the-counter medicines and dietary supplements might contain prohibited substances, and 87% were unaware that names of prohibited substances might not appear on the ingredient labels of dietary supplements. In contrast, attending a lecture on doping was effective in facilitating the acquisition of all these types of knowledge.ConclusionsIt is important to provide more opportunities for appropriate education of pharmacy students on the topic of doping, given that interest exists and attending a lecture on the topic appears to be useful. More education about doping for pharmacy students would be as effective for anti-doping activities as is education of athletes.

Spatiotemporal dynamics of intracellular calcium in the middle cerebral artery isolated from stroke-prone spontaneously hypertensive rats

The spatiotemporal dynamics of intracellular calcium within the middle cerebral artery (MCA) isolated from stroke‐prone spontaneously hypertensive rats (SHR‐SP) were investigated using real‐time confocal laser microscopy. At 3 months of age (prestroke), rhythmical changes in the [Ca2+]i during the tonic phase were found to precede vasomotion following application of 5‐HT, but not other stimuli. These responses were not observed at 1 month of age; moreover, the MCA lost both responses post‐stroke (5 months of age). When [Ca2+]i was analysed in arteriolar smooth muscle cells, rhythmical changes in [Ca2+]i occurred during the same cycle. Thus, these processes were synchronized. The synchronized rhythmical changes in [Ca2+]i were abolished following application of 100 nm ketanserin and 10 μm nicardipine. Treatment with 60 nm charybdotoxin and 10 μm cyclopiazonic acid also significantly reduced rhythmical elevations in [Ca2+]i. In addition, rhythmical changes in [Ca2+]i became unsynchronized following treatment with 100 μm carbenoxolone, a gap junction blocker. Connexin 45 mRNA and protein expression were both elevated in the MCA of SHR‐SP. Taken together, these findings suggest that rhythmical changes in [Ca2+]i of the MCA are dependent upon the 5‐HT2 receptor‐mediated release of calcium from intracellular stores which, in turn, activates voltage‐dependent calcium channels to enable an influx of calcium into smooth muscle cells. Subsequently, charybdotoxin‐sensitive potassium channels are activated and provide a negative feedback pathway to regulate [Ca2+]i. Moreover, the co‐ordinated synchronization of rhythmical changes in [Ca2+]i across smooth muscle cells was found to be dependent upon gap junctions.