MEK inhibitors increase the mortality rate in mice with LPS-induced inflammation through IL-12-NO signaling.

Lipopolysaccharide (LPS) is an endotoxin that can cause an acute inflammatory response. Nitric oxide (NO) is one of the most important innate immune system components and is synthesized by inducible NOS (iNOS) in macrophages in response to stimulation with LPS. LPS activates the RAS-RAF-mitogen-activated protein kinase/ERK kinase (MEK)-extracellular-signal-regulated kinase (ERK) signaling cascade in macrophages. The purpose of this study was to examine how the combination of LPS and MEK inhibitors, which have been used as anticancer agents in recent years, affects inflammation. We showed that MEK inhibitors enhanced iNOS expression and NO production in LPS-stimulated mouse bone marrow-derived macrophages. A MEK inhibitor increased the mortality rate in mice with LPS-induced inflammation. The expression of the cytokine interleukin-12 (IL-12) in macrophages was enhanced by the MEK inhibitor, as shown by a cytokine array and ELISA. IL-12 enhanced iNOS expression and NO production in response to LPS. We also showed that tumor necrosis factor (TNF-α) was secreted by macrophage after stimulation with LPS and that TNF-α and IL-12 synergistically induced iNOS expression and NO production. An anti-IL-12 neutralizing antibody prevented NO production and mortality in an LPS-induced inflammation mouse model in the presence of a MEK inhibitor. These results suggest that the MEK inhibitor increases the mortality rate in mice with LPS-induced inflammation through IL-12-NO signaling.

Long-term glucocorticoid treatment increases CD204 expression by activating the MAPK pathway and enhances modified LDL uptake in murine macrophages.

Atherosclerotic plaques develop from the accumulation of macrophage-derived foam cells via the uptake of modified low-density lipoprotein (LDL). CD36 and CD204 are the principal scavenger receptors responsible for the uptake of modified LDL. Although glucocorticoids are suspected to exacerbate atherosclerosis, the precise mechanisms have not been fully elucidated. We investigated the effects of long-term treatment (2 weeks) with both a natural glucocorticoid (hydrocortisone, HC, 1 µM) and a synthetic glucocorticoid (dexamethasone, Dex, 100 nM) on murine bone marrow-derived macrophages using flow cytometry and western blotting. Treatment with HC and Dex enhanced CD204 expression but not CD36 expression and acetylated LDL (Ac-LDL) uptake. Treatment with HC and Dex also induced the phosphorylation of extracellular signal-regulated kinase (ERK). The Dex-induced enhancement in CD204 expression and Ac-LDL uptake were suppressed by an inhibitor of the mitogen-activated protein kinase (MAPK)/ERK kinase. These results suggest that glucocorticoids activate the MAPK/ERK pathway, which enhances CD204 expression and results in increased uptake of Ac-LDL in macrophages. The MAPK/ERK pathway in macrophages might be a key target to prevent atherosclerosis that is worsened by glucocorticoids.

JAK-STAT-dependent regulation of scavenger receptors in LPS-activated murine macrophages.

In atherosclerosis progression, atherosclerotic plaques develop upon accumulated foam cells derived from macrophages that take up modified low-density lipoprotein (LDL). CD36 and CD204 are the principal scavenger receptors responsible for the uptake of modified LDL. Lipopolysaccharide (LPS) exacerbates atherosclerosis by enhancing the expression of scavenger receptors and thus increasing the uptake of modified LDL into macrophages. However, the signaling pathways that mediate LPS and scavenger receptor expression have not been fully elucidated. We used mouse bone marrow-derived macrophages and investigated the effects of LPS in vitro. LPS enhanced the phosphorylation of extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription-1 (STAT-1). Inhibitors of the mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) pathway (U0126 and PD0325901) suppressed the uptake of acetylated-LDL (Ac-LDL) and the expression of CD204 but not CD36 in LPS-activated macrophages. Inhibitors of the Janus tyrosine kinase (JAK)-STAT pathway (ruxolitinib and tofacitinib) suppressed the uptake of Ac-LDL and the expression of both CD36 and CD204 in LPS-activated macrophages. We next injected LPS into the peritoneal cavity of mice and analyzed the effects of LPS. MEK inhibitor U0126 suppressed the uptake of Ac-LDL and the expression of CD204 but not CD36 in LPS-activated macrophages. JAK inhibitor ruxolitinib suppressed the uptake of Ac-LDL and the expression of both CD36 and CD204 in LPS-activated macrophages. These results suggest that scavenger receptors in LPS-activated mouse macrophages are regulated through a JAK-STAT-dependent pathway. Although further evaluation is necessary, JAK-STAT inhibition could be useful in atherosclerosis therapy, at least for atherosclerosis exacerbated by LPS.

Phorbol 12-myristate 13-acetate (PMA) suppresses high Ca2+-enhanced adipogenesis in bone marrow stromal cells.

We have previously reported that increased extracellular and intracellular Ca2+ lead to adipocyte accumulation in bone marrow stromal cells (BMSCs). However, strategies to suppress high Ca2+-enhanced adipocyte accumulation have not been reported. We examined the effects of the diacylglycerol analog phorbol 12-myristate 13-acetate (PMA) on proliferation and adipogenesis of mouse primary BMSCs. We used 9 mM CaCl2 and 100 nM ionomycin to increase extracellular Ca2+ and intracellular Ca2+, respectively. PMA suppressed the expression of both C/EBPα and PPARγ under normal adipogenesis, adipogenesis + CaCl2, and adipogenesis + ionomycin conditions. PMA enhanced proliferation under normal adipogenesis conditions but suppressed proliferation under adipogenesis + CaCl2 and adipogenesis + ionomycin conditions. PMA did not affect the accumulation of adipocytes under normal adipogenesis conditions but suppressed adipocyte accumulation under adipogenesis + CaCl2 and adipogenesis + ionomycin conditions. These results suggest that the PMA-dependent pathway is an important signaling pathway to suppress high Ca2+-enhanced adipocyte accumulation.

A simple method to increase the proportion of bone marrow-derived macrophages positive for M-CSFR using the reducing agent dithiothreitol (DTT).

Only a few bone marrow-derived macrophages (BM-MΦ) are positive for macrophage colony-stimulating factor receptor (M-CSFR). Thus, a method is needed to increase the proportion of BM-MΦ that are positive for M-CSFR to facilitate the investigation of the effects of M-CSFR downregulation on various diseases. We used mouse primary BM-MΦ to evaluate the expression of M-CSFR on the cytoplasmic membrane using flow cytometry. Treatment with a reducing agent, dithiothreitol (DTT), increased the proportion of BM-MΦ that were positive for M-CSFR, and this increase was time dependent. We next determined whether DTT-treated BM-MΦ can lead to the downregulation of M-CSFR. Treatment with lipopolysaccharide (LPS) for 24 h. decreased the proportion of DTT-treated BM-MΦ that were positive for M-CSFR. These results suggest that DTT treatment increases the proportion of BM-MΦ that are positive for M-CSFR and that the upregulation of M-CSFR on BM-MΦ can be abrogated by treatment with LPS. Here, we propose a simple method to increase the number of M-CSFR-positive BM-MΦ using the reducing agent DTT, which could be useful in investigations of the relationship between the downregulation of M-CSFR and some diseases. •The proportion of BM-MΦ that expresses M-CSFR on the membrane increases by approximately twice following DTT treatment.

LPS enhances expression of CD204 through the MAPK/ERK pathway in murine bone marrow macrophages.

BACKGROUND AND AIMS: Lipopolysaccharide (LPS) is a main component of the Gram-negative bacterial cell wall and is associated with a greater risk of atherosclerosis development in periodontal disease. LPS has been reported to increase both CD36 and CD204 expression and enhance the uptake of modified low-density lipoprotein (LDL). However, the signaling pathways by which LPS enhances these expression levels and function have not been fully elucidated, although the clarification of these signaling pathways is important for identifying therapeutic targets for atherosclerosis. METHODS AND RESULTS: We have shown here that LPS activated the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway, increased both CD204 and CD36 expression, and enhanced the uptake of acetylated-LDL (Ac-LDL) in mouse bone marrow macrophages. The MAPK/ERK kinase (MEK) inhibitors, U0126 (1 µM) and PD0325901 (10 nM), did not affect the expression of either CD36 or CD204 or the uptake of Ac-LDL under normal conditions (no treatment with LPS). In contrast, U0126 (1 µM) and PD0325901 (10 nM) blocked the LPS-induced increase in Ac-LDL uptake and CD204 expression but not CD36 expression. CONCLUSIONS: These results suggest that LPS may increase Ac-LDL uptake and enhance CD204 expression through MAPK/ERK activation and CD36 expression through an ERK-independent pathway. Since MEK inhibitors block CD204 expression in mouse BM macrophages only under LPS treatment but not under normal conditions, a MEK inhibitor might be a good candidate compound for the treatment of LPS-induced atherosclerosis.

High extracellular Ca2+ enhances the adipocyte accumulation of bone marrow stromal cells through a decrease in cAMP.

Bone marrow stromal cells (BMSCs) are common progenitors of both adipocytes and osteoblasts. We recently suggested that increased [Ca2+]o caused by bone resorption might accelerate adipocyte accumulation in response to treatment with both insulin and dexamethasone. In this study, we investigated the mechanism by which high [Ca2+]o enhances adipocyte accumulation. We used primary mouse BMSCs and evaluated the levels of adipocyte accumulation by measuring Oil Red O staining. CaSR agonists (both Ca2+ and Sr2+) enhanced the accumulation of adipocytes among BMSCs in response to treatment with both insulin and dexamethasone. We showed that high [Ca2+]o decreases the concentration of cAMP using ELISA. Real-time RT-PCR revealed that increasing the intracellular concentration of cAMP (both chemical inducer (1µM forskolin and 200nM IBMX) and a cAMP analog (10µM pCPT-cAMP)) suppressed the expression of PPARγ and C/EBPα. In addition, forskolin, IBMX, and pCPT-cAMP inhibited the enhancement in adipocyte accumulation under high [Ca2+]o in BMSCs. However, this inhibited effect was not observed in BMSCs that were cultured in a basal concentration of [Ca2+]o. We next observed that the accumulation of adipocytes in the of bone marrow of middle-aged mice (25-40 weeks old) is higher than that of young mice (6 weeks old) based on micro CT. ELISA results revealed that the concentration of cAMP in the bone marrow mononuclear cells of middle-aged mice is lower than that of young mice. These data suggest that increased [Ca2+]o caused by bone resorption might accelerate adipocyte accumulation through CaSR following a decrease in cAMP.

Increased extracellular and intracellular Ca²âº lead to adipocyte accumulation in bone marrow stromal cells by different mechanisms.

Mesenchymal stem cells found in bone marrow stromal cells (BMSCs) are the common progenitors for both adipocyte and osteoblast. An increase in marrow adipogenesis is associated with age-related osteopenia and anemia. Both extracellular and intracellular Ca(2+) ([Ca(2+)]o and [Ca(2+)]i) are versatile signaling molecules that are involved in the regulation of cell functions, including proliferation and differentiation. We have recently reported that upon treatment of BMSCs with insulin and dexamethasone, both high [Ca(2+)]o and high [Ca(2+)]i enhanced adipocyte accumulation, which suggested that increases in [Ca(2+)]o caused by bone resorption may accelerate adipocyte accumulation in aging and diabetic patients. In this study, we used primary mouse BMSCs to investigate the mechanisms by which high [Ca(2+)]o and high [Ca(2+)]i may enhance adipocyte accumulation. In the process of adipocyte accumulation, two important keys are adipocyte differentiation and the proliferation of BMSCs, which have the potential to differentiate into adipocytes. Use of MTT assay and real-time RT-PCR revealed that high [Ca(2+)]i (ionomycin)-dependent adipocyte accumulation is caused by enhanced proliferation of BMSCs but not enhanced differentiation into adipocytes. Using fura-2 fluorescence-based approaches, we showed that high [Ca(2+)]o (addition of CaCl2) leads to increases in [Ca(2+)]i. Flow cytometric methods revealed that high [Ca(2+)]o suppressed the phosphorylation of ERK independently of intracellular Ca(2+). The inhibition of ERK by U0126 and PD0325901 enhanced the differentiation of BMSCs into adipocytes. These data suggest that increased extracellular Ca(2+) provides the differentiation of BMSCs into adipocytes by the suppression of ERK activity independently of increased intracellular Ca(2+), which results in BMSC proliferation.

Enhanced accumulation of adipocytes in bone marrow stromal cells in the presence of increased extracellular and intracellular [Ca²âº].

The bone marrow stroma contains osteoblasts and adipocytes that have a common precursor: the pluripotent mesenchymal stem cell found in bone marrow stromal cells (BMSCs). Local bone marrow Ca(2+) levels can reach high concentrations due to bone resorption, which is one of the notable features of the bone marrow stroma. Here, we describe the effects of high [Ca(2+)](o) on the accumulation of adipocytes in the bone marrow stroma. Using primary mouse BMSCs, we evaluated the level of adipocyte accumulation by measuring Oil Red O staining and glycerol-3-phosphate dehydrogenase (GPDH) activity. High [Ca(2+)](o) enhanced the accumulation of adipocytes following treatment with both insulin and dexamethasone together but not in the absence of this treatment. This enhanced accumulation was the result of both the accelerated proliferation of BMSCs and their differentiation into adipocytes. Using the fura-2 method, we also showed that high [Ca(2+)](o) induces an increase in [Ca(2+)](i). An intracellular Ca(2+) chelator suppressed the enhancement in adipocyte accumulation due to increased [Ca(2+)](o) in BMSCs. These data suggest a new role for extracellular Ca(2+) in the bone marrow stroma: increased [Ca(2+)](o) induces an increase in [Ca(2+)](i) levels, which in turn enhances the accumulation of adipocytes under certain conditions.

T-type ca(2+) channel blockers increase smooth muscle progenitor cells and endothelial progenitor cells in bone marrow stromal cells in culture by suppression of cell death.

OBJECTIVE: To examine the expression patterns and roles of voltage-dependent Ca2+ channels in bone marrow stromal cells (BMSCs). MATERIALS AND METHODS: Ca(2+) currents of BMSCs were measured by the whole-cell patch clamp method. The number and percentage of deaths of BMSCs cultured for 14 days with or without Ca(2+) channel blockers were evaluated using a MTT assay and an LDH assay, respectively. RESULTS: T-type Ca(2+) channel current was recorded in 0, 2, 10, and 4% of BMSCs on days 3, 10, 17, and 24 in culture, respectively. L-type Ca(2+) channel current was first recorded on day 24 in 6% of BMSCs. Addition of the T-type Ca(2+) channel blocker mibefradil but not the L-type Ca(2+) channel blocker nifedipine significantly increased the cell count. Immunocytochemical analysis revealed increases in the counts of smooth muscle progenitor cells (SMPCs) and endothelial progenitor cells (EPCs). Mibefradil but not nifedipine significantly decreased the rate of cell death. CONCLUSION: T-type Ca(2+) channel blockers increased the numbers of SMPCs and EPCs in cultured BMSCs, partly through suppression of cell death. Thus, T-type Ca(2+) channel blockers may have the potential to provide an increased number of both BMSC-derived SMCs and ECs of potential use in cell and gene therapy.

Acute myocardial infarction with myocardial perfusion defect detected by contrast-enhanced computed tomography.

The first case was a 68-year-old woman who had acute migratory pain from back to anterior chest and the second case was 66-year-old man with a cardiac tamponade. Two cases were demonstrated with a low density area of the left ventricular postero-lateral wall with conventional contrast-enhanced computed tomography (CE-CT) performed to differentiate the diagnosis of acute coronary syndrome and acute aortic dissection. Subsequent coronary angiograms showed the lesions of left circumflex. These cases of early contrast-defect corresponded to a decreased myocardial blood flow with AMI. CE-CT image facilitated the diagnosis of AMI preceding CAG examination.

Platelets activated by collagen through the immunoreceptor tyrosine-based activation motif in the Fc receptor gamma-chain play a pivotal role in the development of myocardial ischemia-reperfusion injury.

Platelet activation and the formation of platelet microaggregates in coronary vessels play pivotal roles in myocardial ischemia and reperfusion injury. The Fc receptor gamma-chain (FcR gamma) is coexpressed with glycoprotein (GP) VI, forming a platelet collagen receptor, and the activation of platelets by collagen is closely coupled with tyrosine phosphorylation of the FcRgamma. To examine the functional significance of platelet FcR gamma/GPVI complex in the early phase of myocardial ischemia and reperfusion injury in mice, we performed coronary occlusion and reperfusion experiments using wild type mice and FcRgamma-deficient (FcRgamma(-/-)) mice that lack GPVI. The infarct size was significantly smaller in FcRgamma(-/-) mice subjected to occlusion and reperfusion of the coronary artery than in control FcR gamma(+/+) mice. Twenty-four hours after the reperfusion, electron microscopy of the injured tissue showed substantially more platelet aggregation and occlusive platelet microthrombi in the capillaries of the damaged areas of the wild type mice than in those of the FcR gamma(-/-) mice. Platelet Syk was scarcely activated in the FcR gamma(-/-) mice after myocardial ischemia and reperfusion, but significantly activated in the FcR gamma(+/+) mice. CD11b expression on neutrophils was elevated after myocardial ischemia and reperfusion in both mouse groups, whereas myeloperoxidase activity in the injured areas was significantly lower in the FcRgamma(-/-) mice than in the FcRgamma(+/+) mice. These results suggest that the collagen-induced activation of platelets through the FcR gamma plays a pivotal role in the extension of myocardial ischemia-reperfusion injury. FcRgamma and GPVI may be important therapeutic targets for myocardial ischemia-reperfusion injury.

Isolation of bone marrow stromal cell-derived smooth muscle cells by a human SM22alpha promoter: in vitro differentiation of putative smooth muscle progenitor cells of bone marrow.

BACKGROUND: Bone marrow stromal cells (BMSCs) have many characteristics of mesenchymal stem cells that can differentiate into smooth muscle cells (SMCs). However, there have been few studies closely following the cell development of smooth muscle lineage among BMSCs. METHODS AND RESULTS: To investigate the possible existence of a cell population committed to the SMC lineage among bone marrow adhesion cells, we tried to detect and follow the in vitro differentiation of such a cell type by using a promoter-sorting method with a human SM22alpha promoter (-480 bp)/green fluorescent protein (GFP) construct. The construct was transfected to adhesion cells that appeared 5 days after the seeding of mononuclear cells from bone marrow. GFP was first detectable 5 days after the transfection in a cell population [Ad(G) cells], which expressed PDGF-beta but neither mature (calponin) nor immature (SMemb) SMC-specific proteins at that time. However, the cells were eventually grown into individual clones that expressed SMC-specific proteins (alpha-smooth muscle actin, calponin, and SM-1), suggesting that Ad(G) cells have partly at least progenitor properties. Because early studies have reported that PDGF-beta signaling plays pivotal roles in the differentiation of mesenchymal smooth muscle progenitor cells, Ad(G) cells might be putative mesenchymal smooth muscle progenitors expressing PDGF-beta. CONCLUSIONS: We demonstrated the presence of a cell population fated to become SMCs and followed their differentiation into SMCs among BMSCs.

Platelets activated by collagen through immunoreceptor tyrosine-based activation motif play pivotal role in initiation and generation of neointimal hyperplasia after vascular injury.

BACKGROUND: Platelet adhesion on components of the extracellular matrix and platelet activation by those components are crucial for the arrest of posttraumatic bleeding, but they can also harm tissue by occluding diseased vessels. Recent studies have shown that the activation of platelets by collagen is mediated through the same pathway used by immune receptors, with an immunoreceptor tyrosine-based activation motif on the Fc receptor gamma chain (FcRgamma) playing a pivotal role. METHODS AND RESULTS: We examined the role of collagen-stimulated platelets in the development of injury-induced neointimal formation by using mice deficient in FcRgamma. The left femoral arteries of 8- to 12-week-old FcRgamma-deficient mice (n=16) and C57BL/6 (wild-type) mice (n=16) were injured by a straight spring wire (0.35-mm diameter). Segments of the injured and uninjured femoral arteries were excised at 7 days and 28 days after the vascular injury. Arterial segments were examined by immunohistochemistry and electron microscopy. Two hours after injury, electron microscopy showed marked decreases in platelet adhesion and neutrophil attachment to the vascular wall surface in FcRgamma-knockout mice compared with wild-type mice. At 7 days after injury, staining with anti-neutrophil antibody showed fewer neutrophils in FcRgamma-knockout mice than in wild-type mice. Computer-aided morphometry performed to measure the neointimal area, intima/media ratio, and stenotic area at 28 days after injury showed a significantly smaller ratio and area in FcRgamma-knockout mice than in wild-type mice (for neointimal area, 16 635 +/- 1406 versus 31 483 +/- 2309 microm2, respectively; for intima/media ratio, 1.25 +/- 0.40 versus 2.68 +/- 0.04, respectively; and for stenotic area, 26.8 +/- 2.1% versus 49.3 +/- 4.1%, respectively). CONCLUSIONS: These results demonstrate that FcRgamma may play important roles in the initiation and generation of neointimal hyperplasia after balloon injury through the activation of platelets by collagen.