Keiichi Nakajima

Simvastatin enhances the regeneration of endothelial cells via VEGF secretion in injured arteries.

The search for a novel therapy for endothelial regenerating is an area of intensive investigation. Recent experimental and clinical evidence strongly suggests that 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors (statins) have several physiological effects independent of low-density lipoprotein cholesterol reduction. We here report that the carotid arterial blood flow after endothelial injury in hamsters treated with simvastatin was restored, in contrast to the situation in nontreated hamsters. Histologic observations showed a prompt recovery of endothelial cells with a much higher DNA synthesis index in repaired endothelium of hamsters treated with simvastatin. The amount of secreted vascular endothelial cell growth factor (VEGF) by cultured vascular smooth muscle cells from hamsters treated with simvastatin was significantly increased. Mevalonate reduced the amount of VEGF secretion by simvastatin in vitro. Finally, an injection of either an anti-VEGF antibody or an anti-VEGF receptor-1 (Flt-1) antibody, but not anti-VEGF receptor-2 (Flk-1), reduced the prompt endothelial healing. Simvastatin regulates endothelial regenerating by an over-release of VEGF and by this may result in prompt endothelial healing after vascular injury. Our results provide new insights into the role of statin and VEGF in the pathogenesis of vascular diseases.

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.

Plasminogen/Plasmin Modulates Bone Metabolism by Regulating the Osteoblast and Osteoclast Function

The contribution of plasminogen (Plg)/plasmin, which have claimed to be the main fibrinolytic regulators in the bone metabolism, remains unclear. This study evaluated how the absence of Plg affects the function of osteoblast (OB) and osteoclast (OC). There was a larger population of pre-OCs in bone marrow-derived cells from the Plg−/− mice than the population of that from the WT mice. In addition, the absence of Plg suppressed the expression of osteoprotegerin in OBs. Moreover, an exogenous plasmin clearly induced the osteoprotegerin expression in Plg−/− OBs. The osteoclastogenesis of RAW264.7 mouse monocyte/macrophage lineage cells in co-culture with OBs from the Plg−/− mice was significantly accelerated in comparison with that in co-culture with OBs from the WT mice. Intriguingly, the accelerated OC differentiation of RAW264.7 cells co-cultured with Plg−/− OBs was clearly suppressed by the treatment of an exogenous plasmin. Consequently, Plg−/− mice display decreased bone mineral density. These findings could eventually lead to the development of new clinical therapies for bone disease caused by a disorder of the fibrinolytic system.

Bovine milk lactoferrin induces synthesis of the angiogenic factors VEGF and FGF2 in osteoblasts via the p44/p42 MAP kinase pathway

Lactoferrin (LF) belongs to the transferrin family and is present in several physiological fluids, including milk and colostrum. LF has recently been identified as an anabolic factor for bone. Here we investigated whether bovine LF (bLF) induces synthesis of angiogenic factors by osteoblasts. If so, we examined the underlying mechanism. We found that bLF purified from milk increased the mRNA expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) in murine osteoblast-like MC3T3-E1 cells and primary murine osteoblasts in a time- and dose-dependent manner. Furthermore, bLF increased VEGF and FGF2 protein levels in MC3T3-E1 cells. In addition, treatment of MC3T3-E1 cells with bLF rapidly induced phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase. The bLF-mediated increases in VEGF and FGF2 mRNA and protein were inhibited by U0126, a specific inhibitor of the upstream kinase that activates p44/p42 MAP kinase (MEK). Taken together, our results strongly suggest that bLF induces VEGF and FGF2 synthesis in a p44/p42 MAP kinase-dependent manner in MC3T3-E1 cells.

Adenylyl cyclase-cAMP system inhibits thyroid hormone-stimulated osteocalcin synthesis in osteoblasts

It is generally recognized that thyroid hormone modulates osteoblast cell function. We have previously shown that triiodothyronine (T(3)) activates p38 mitogen-activated protein (MAP) kinase, resulting in the synthesis of osteocalcin in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effect of the adenylyl cyclase-cAMP system on thyroid hormone-stimulated osteocalcin synthesis in these cells. Dibutyryl-cAMP (DBcAMP) reduced the osteocalcin synthesis stimulated by T(3). Forskolin and cholera toxin suppressed the osteocalcin synthesis while dideoxyforskolin, a forskolin derivative that does not activate adenylyl cyclase, had little effect on the synthesis. KT5720, a selective inhibitor of protein kinase A, reversed the inhibitory effect of forskolin or DBcAMP. DBcAMP and forskolin markedly reduced the phosphorylation of p38 MAP stimulated by T(3). Pituitary adenylate cyclase-activating polypeptide (PACAP) significantly inhibited the T(3)-stimulated osteocalcin synthesis. These results strongly suggest that the adenylyl cyclase-cAMP system has an inhibitory role in thyroid hormone-stimulated osteocalcin synthesis via suppression of p38 MAP kinase activation in osteoblasts.

A peptide isolated from αB‐crystallin is a novel and potent inhibitor of platelet aggregation via dual prevention of PAR‐1 and GPIb/V/IX

Summary.  Background: The ability of low‐molecular‐weight heat shock protein (HSP) to modulate thrombin‐induced platelet aggregation has been investigated. Objectives: We examined the inhibitory effects on platelet aggregation of nine amino acid sequences isolated from HSP20 or αB‐crystallin and their various derivatives. Methods and results: Platelet aggregation induced by various agonists was performed. These findings indicated that a peptide (Trp‐Ile‐Arg‐Arg‐Pro‐Phe‐Phe‐Pro‐Phe) from αB‐crystallin significantly inhibits platelet aggregation induced by thrombin, TRAP (a protease activated receptor‐1 agonist) and botrocetin, ristocetin (a stimulator of the platelet glycoprotein Ib/V/IX–von Willebrand factor axis), but not a protease‐activated receptor‐4 agonist, collagen and ADP. The inhibitory activity against thrombin or botrocetin is mainly linked to Arg‐Arg‐Pro‐Phe or Trp‐Ile‐Arg‐Arg‐Pro, respectively, among nine amino acids. Additionally, during in vivo experiments, Trp‐Ile‐Arg‐Arg‐Pro‐Phe‐Phe‐Pro‐Phe shows a significant antithrombotic effect without marked bleeding. Conclusion: Our results provide the basis for a potential new aspect of antiplatelet compound for the therapy of thrombosis and cardiovascular disease.

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.

Possible Involvement of Prolactin in the Synthesis of Lactoferrin in Bovine Mammary Epithelial Cells

Bovine mammary epithelial cells (bMECs) synthesize lactoferrin, which is secreted into milk. Our results suggest that prolactin stimulated secretion of lactoferrin in primary bMECs and their clonal cell line under serum-free conditions. Prolactin also stimulated mRNA expression of lactoferrin in the clonal cell line. This effect was reduced by AG-490, suggesting that the prolactin-stimulated mRNA expression of lactoferrin was mediated by Janus kinase (JAK)2.

Involvement of SAPK/JNK in prostaglandin E1-induced VEGF synthesis in osteoblast-like cells

We previously reported that prostaglandin E(1) (PGE(1)) activates both p44/p42 mitogen-activated protein (MAP) kinase and p38 MAP kinase via cAMP-dependent protein kinase in osteoblast-like MC3T3-E1 cells, and that p38 MAP kinase but not p42/p44 MAP kinase is involved in PGE(1)-induced synthesis of vascular endothelial growth factor (VEGF). In the present study, we investigated the involvement of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in the PGE(1)-induced VEGF synthesis in MC3T3-E1 cells. PGE(1) induced the phosphorylation of SAPK/JNK. SP600125, a specific inhibitor of SAPK/JNK, markedly reduced the PGE(1)-induced VEGF synthesis. Forskolin, a direct activator of adenylyl cyclase, elicited the phosphorylation of SAPK/JNK, and 8bromo-cAMP, a plasma membrane-permeable cAMP analogue-stimulated VEGF synthesis was significantly reduced by SP600125. SP600125 suppressed the PGE(1)-induced phosphorylation of SAPK/JNK without affecting the phosphorylation of p38 MAP kinase induced by PGE(1). The phosphorylation of c-Jun induced by PGE(1) was also inhibited by SP600125. SB203580, a p38 MAP kinase inhibitor, failed to reduce the PGE(1) induced phosphorylation of SAPK/JNK. A combination of SP600125 and SB203580 suppressed the PGE(1)-stimulated VEGF synthesis in an additive manner. These results strongly suggest that PGE(1) activates SAPK/JNK in osteoblasts, and that SAPK/JNK plays a part in PGE(1)-induced VEGF synthesis.

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.