Kouseki Hirade

Mechanism of simvastatin on induction of heat shock protein in osteoblasts

It has recently been reported that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) stimulate bone formation. However, the mechanism of stimulation of bone metabolism by statins is not precisely clarified. In this study, we investigated whether simvastatin induces heat shock protein (HSP) 27, HSP70, and HSP90 in osteoblast-like MC3T3-E1 cells. Simvastatin increased the levels of HSP27 while having little effect on the levels of HSP70 or HSP90. The effect of simvastatin on HSP27 accumulation was dose dependent. Cycloheximide reduced the accumulation. Simvastatin induced an increase in the levels of mRNA for HSP27. Actinomycin D suppressed the mRNA levels. Simvastatin induced the phosphorylation of p38 mitogen-activated protein (MAP) kinase among the MAP kinase superfamily. SB203580 and PD169316, inhibitors of p38 MAP kinase, suppressed the HSP27 accumulation by simvastatin while SB202474, a negative control of p38 MAP kinase inhibitor, had no effect. SB203580 reduced the simvastatin-increased mRNA levels for HSP27. Lovastatin, another statin, also induced the HSP27 accumulation and SB203580 suppressed the HSP27 accumulation. These results strongly suggest that statins such as simvastatin do not stimulate the induction of HSP70 and HSP90, but do stimulate the induction of HSP27 in osteoblasts and that p38 MAP kinase plays a role in this induction.

High-performance liquid chromatographic assay of zonisamide in human plasma using a non-porous silica column.

A new method for measuring zonisamide (ZNS) in plasma by high-performance liquid chromatography was developed by using a 2-microm reversed-phase non-porous silica column. ZNS in plasma was first purified with a column extraction technique and injected onto the non-porous silica column. Calibration curve was linear over the concentration range of 1-80 microg/ml in plasma. The recoveries of ZNS added to plasma were more than 95.4% with the coefficient of variation less than 9.0%. We developed a rapid routine method using the non-porous silica column that was accurate and improved solvent consumption in the measurement of ZNS.

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.

Lack of alpha2-antiplasmin improves cutaneous wound healing via over-released vascular endothelial growth factor-induced angiogenesis in wound lesions.

Summary.  Background: The fibrinolytic system is supposed to play an important role in the degradation of extracellular matrices for physiological and pathological tissue remodeling; however, the detailed mechanism regarding how this system affects cutaneous wound healing remains to be clarified. Methods and results: We performed experimental cutaneous wounding in mice with a deficiency of α2‐antiplasmin (α2AP), which is a potent and specific plasmin inhibitor. We found that an accelerated wound closure was observed in α2AP‐deficient (α2AP−/−) mice in comparison with wild type (WT) mice. Moreover, we observed that a greater increase of angiogenesis occurred in the process of wound healing in α2AP−/− mice than in the WT mice. Intriguingly, mRNA expression of vascular endothelial growth factor (VEGF), which is the best characterized positive regulator of angiogenesis, in wound lesions was found to show a greater increase in the early phase of the healing process in α2AP−/− mice than in WT mice. In addition, the amount of released‐VEGF from the explanted fibroblasts of α2AP−/− mice increased dramatically more than in the WT mice. Finally, the intra‐jugular administration of anti‐VEGF antibody clearly suppressed the increased angiogenesis and accelerated wound closure in the wound lesion of α2AP−/− mice. Conclusion: The lack of α2AP markedly causes an over‐release of VEGF from the fibroblasts in cutaneous wound lesions, thereby inducing angiogenesis around the area, and thus resulting in an accelerated‐wound closure. Conclusions: This is the first report to describe the crucial role that α2AP plays following angiogenesis in the process of wound healing. Our results provide new insight into the role of α2AP on cutaneous wound healing.

Stress-activated protein kinase/c-Jun N-terminal kinase (JNK) plays a part in endothelin-1-induced vascular endothelial growth factor synthesis in osteoblasts.

We previously reported that endothelin‐1 (ET‐1) activates both p44/p42 mitogen‐activated protein (MAP) kinase and p38 MAP kinase in osteoblast‐like MC3T3‐E1 cells, and that not p44/p42 MAP kinase but p38 MAP kinase participates in the ET‐1‐induced vascular endothelial growth factor (VEGF) synthesis. In the present study, we investigated the involvement of stress‐activated protein kinase/c‐Jun N‐terminal kinase (JNK) in ET‐1‐induced VEGF synthesis in these cells. ET‐1 significantly induced the phosphorylation of JNK in a dose‐dependent manner in the range between 0.1 and 100 nM. SP600125, an inhibitor of JNK, markedly reduced the ET‐1‐induced VEGF synthesis. A combination of SP600125 and SB203580 additively reduced the ET‐1‐stimulated VEGF synthesis. SP600125 suppressed the ET‐1‐induced phosphorylation of JNK, while having no effect on the phosphorylation of p38 MAP kinase elicited by ET‐1. SB203580, an inhibitor of p38 MAP kinase, hardly affected the ET‐1‐induced phosphorylation of JNK. These results strongly suggest that JNK plays a role in ET‐1‐induced VEGF synthesis in addition to p38 MAP kinase in osteoblasts. J. Cell. Biochem. 87: 417–423, 2002.

Activation of p38 mitogen-activated protein kinase mediates thyroid hormone-stimulated osteocalcin synthesis in osteoblasts.

It is well known that thyroid hormone modulates osteoblast cell function. We have previously shown that triiodothyronine (T(3)) activates p44/p42 mitogen-activated protein (MAP) kinase, which limits T(3)-induced alkaline phosphatase activity in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether p44/p42 MAP kinase or p38 MAP kinase is involved in the thyroid hormone-stimulated osteocalcin synthesis in these cells. T(3) markedly induced the phosphorylation of p38 MAP kinase in addition to p44/p42 MAP kinase. PD98059 and U0126, inhibitors of the upstream kinase that activates p44/p42 MAP kinase, had little effect on the T(3)-induced synthesis of osteocalcin. On the contrary, the T(3)-induced osteocalcin synthesis was significantly reduced by SB203580 and PD169316, inhibitors of p38 MAP kinase. SB203580, PD169316 or PD98059 suppressed the T(3)-phosphorylation of myelin basic protein. T(3)-induced osteocalcin synthesis was significantly reduced by SB203580 or PD169316 also in primary cultured mouse osteoblasts. These results strongly suggest that p38 MAP kinase but not p44/p42 MAP kinase takes part in the thyroid hormone-stimulated osteocalcin synthesis in osteoblasts.

Vasopressin phosphorylates HSP27 in aortic smooth muscle cells

Administration of arginine vasopressin (AVP) time‐dependently induced the phosphorylation of heat shock protein 27 (HSP27) at Ser‐15 and Ser‐85 in smooth muscle of aorta in vivo. The AVP‐induced phosphorylation of HSP27 at Ser‐15 and Ser‐85 was inhibited by a V1a receptor antagonist but not by a V2 receptor antagonist. In cultured aortic smooth muscle A10 cells, AVP markedly stimulated the phosphorylation of HSP27 at Ser‐15 and Ser‐85. The AVP‐induced phosphorylation of HSP27 was attenuated by SB203580 and PD169316, inhibitors of p38 mitogen‐activated protein (MAP) kinase, but not by PD98059, a MEK inhibitor. These results strongly suggest that AVP phosphorylates HSP27 via p38 MAP kinase in aortic smooth muscle cells.

Modulation by the steroid/thyroid hormone superfamily of TGF‐β‐stimulated VEGF release from vascular smooth muscle cells

We previously reported that transforming growth factor‐β (TGF‐β) stimulates the release of vascular endothelial growth factor (VEGF) from aortic smooth muscle A10 cells via activation of p38 mitogen‐activated protein (MAP) kinase. In the present study, we investigated whether nuclear hormone receptor superfamily members affect TGF‐β‐stimulated VEGF release from A10 cells. Retinoic acid or 1,25‐dihydroxyvitamin D3 enhanced TGF‐β‐induced VEGF release in a concentration‐dependent manner, whereas dexamethasone or corticosterone suppressed TGF‐β‐induced VEGF release. 1,25‐Dihydroxyvitamin D3 and TGF‐β stimulated phosphorylation of p38 MAP kinase in an additive manner. SB203580, an inhibitor of p38 MAP kinase, decreased the VEGF release induced by TGF‐β or 1,25‐dihydroxyvitamin D3. However, retinoic acid, dexamethasone, or corticosterone did not affect phosphorylation of p38 MAP kinase. These results indicate that retinoic acid, 1,25‐dihydroxyvitamin D3, and glucocorticoids affect TGF‐β‐stimulated VEGF release from aortic smooth muscle cells. The stimulatory effect of 1,25‐dihydroxyvitamin D3 occurs, in part, via modification of TGF‐β‐induced activation of p38 MAP kinase. J. Cell. Biochem.

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.

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.