Erythropoietin, a putative neurotransmitter during hypoxia, is produced in RVLM neurons and activates them in neonatal Wistar rats.

Recent studies indicate that erythropoietin (EPO) is present in many areas of the brain and is active in the restoration of impaired neurons. In this study, we examined the presence of EPO and its role in bulbospinal neurons in the rostral ventrolateral medulla (RVLM). Hypoxia is often accompanied by a high blood pressure (BP). We hypothesized that EPO is produced in response to hypoxia in RVLM neurons and then activates them. To investigate whether RVLM neurons are sensitive to EPO, we examined the changes in the membrane potentials (MPs) of bulbospinal RVLM neurons using the whole cell patch-clamp technique during superfusion with EPO. A brainstem-spinal cord preparation was used for the experiments. EPO depolarized the RVLM neurons, and soluble erythropoietin receptor (SEPOR), an antagonist of EPO, hyperpolarized them. Furthermore, hypoxia-depolarized RVLM neurons were significantly hyperpolarized by SEPOR. In histological examinations, the EPO-depolarized RVLM neurons showed the presence of EPO receptor (EPOR). The RVLM neurons that possessed EPORs showed the presence of EPO and hypoxia-inducible factor (HIF)-2α. We also examined the levels of HIF-2α and EPO messenger RNA (mRNA) in the ventral sites of the medullas (containing RVLM areas) in response to hypoxia. The levels of HIF-2α and EPO mRNA in the hypoxia group were significantly greater than those in the control group. These results suggest that EPO is produced in response to hypoxia in RVLM neurons and causes a high BP via the stimulation of those neurons. EPO may be one of the neurotransmitters produced by RVLM neurons during hypoxia.

Clinical benefits of bortezomib-containing regimens for newly diagnosed AL amyloidosis with severe cardiac impairment.

Cardiac amyloid light-chain amyloidosis (AL amyloidosis) is a rare disease with a very poor prognosis, associated with plasma cell dyscrasias such as monoclonal gammopathy of undetermined significance and multiple myeloma. Though bortezomib-containing regimens have achieved high hematologic response rates, there are still few reports describing the outcomes of Japanese patients. Six patients with severe cardiac AL amyloidosis were treated with bortezomib-containing regimens. Involved free light chain (iFLC) decreased immediately in most of these cases. However, the condition of heart failure and N-terminal pro-B-type natriuretic peptide (NT-proBNP) worsened in the early phase of this treatment and then improved several months later. At 29 months, the median duration of follow-up (2-47months), all patients remain alive except one who died of sudden cardiac arrest. Bortezomib-containing regimens are considered to be among the effective treatments for severe cardiac AL amyloidosis.

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.

Bradyarrhythmias may induce central sleep apnea in a patient with obstructive sleep apnea.

The relationship between central sleep apnea (CSA) and bradyarrhythmia remains unclear. We report the case of a 70-year-old man with severe obstructive sleep apnea and bradyarrhythmia due to sick sinus syndrome in whom concomitant CSA was alleviated after pacemaker implantation.

Suspected panosteitis in a crossbred calf.

A male crossbred calf developed a limp and pain upon deep pressure on the right hind limb and the right forelimb. The radiographic findings of affected limbs and pathological findings of bone biopsy were similar to those observed in canine panosteitis. This is the first case of suspected panosteitis reported in cattle.

Panostéite suspectée chez un veau de race croisée. Un veau mâle de race croisée a développé une boiterie et de la douleur à l'application d'une pression profonde sur la jambe arrière droite et la jambe avant droite. Les résultats de la radiographie des membres touchés et les résultats pathologiques d'une biopsie osseuse étaient semblables à ceux observés dans la panostéite canine. Il s'agit du premier cas de panostéite suspectée chez le bétail.(Traduit par Isabelle Vallières).

Protective efficacy of Babesia gibsoni culture-derived exoantigens against the challenge infection in dogs.

The aim of this study is to determine the efficacy of exoantigens derived from Babesia gibsoni cultures to induce protective immunity against challenge exposure of virulent organisms. An attenuated B. gibsoni Oita strain was maintained in vitro by the microaerophilus stationary phase (MASP) method, and exoantigens-containing supernatant fluids were collected for preparation of the immunization. Two dogs received three subcutaneous immunizations with a 20-day interval of B. gibsoni exoantigens plus 0.5 mg saponin (Quil A). On day 68 after the prime immunization, the immunized dogs and control dogs were challenged intravenously with 2 × 10(8) virulent parasites of a homologous B. gibsoni strain. The results showed that exoantigens could induce a high degree of protection against virulent homologous challenge exposure. Two dogs immunized with exoantigens showed a lower parasitemia, accompanied by a slight decrease in the PCV that returned to normal values. Control dogs developed typical acute clinical signs, including severe anemia and hyperthermia. The immunization elicited humoral immune responses. In dogs immunized with exoantigens, the maximum antibody titer was 2,560 and 5,120 by indirect fluorescent antibody test (IFAT), respectively. Preliminary Western blot analysis of the immunogen revealed five dominant proteins of molecular weights of 18, 37, 43, 50, and 57 kDa. These results suggested that the culture-derived exoantigens were candidates for non-viable vaccine.

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.

An efficient protocol for inducing pseudopregnancy using estradiol dipropionate and follicular development associated with changes in reproductive hormones after prostaglandin F2alpha treatment in pseudopregnant sows.

BACKGROUND: Utilization of estrus synchronization program in livestock industry would provide greater options for reproductive management in herd. To develop a convenient method for estrus synchronization in pigs, we determined the effective protocol using estradiol dipropionate (EDP) for the establishment of pseudopregnancy and investigated follicular development during the estrus synchronization with prostaglandin F2alpha (PGF2alpha) in association with reproductive hormone profiles in pseudopregnant sows. METHODS: In Experiment 1, the effective dose (0, 10, 20, or 30 mg) and timing (5, 8, 11 or 13 days after ovulation) of a single administration of EDP in cyclic pigs for the induction of pseudopregnancy were investigated. In Experiment 2, four pseudopregnant sows were treated with PGF2alpha twice at a 24-h interval between 24 and 28 days after EDP treatment. The changes in plasma concentrations of reproductive hormones were analyzed by time-resolved fluoroimmunoassay. Follicular development and ovulation following PGF2alpha administration were monitored by transrectal ultrasonography. RESULTS: High efficiency (greater than 80%) of pseudopregnancy was achieved with a single treatment with 20 mg of EDP at 8 and 11 days after ovulation (equivalent to 9-13 days after the onset of estrus). Plasma estradiol-17beta concentrations in pseudopregnant sows were significantly higher between 12 h and 7 days than before EDP treatment. Total inhibin concentrations significantly decreased following EDP treatment and remained low for 14 days. The number of small follicles was increased from 6.3 +/- 2.6 at PGF2alpha treatment to 22.8 +/- 4.8 at 3 days later; this was associated with increased plasma concentrations of inhibin. Onset of estrus was detectable in all sows on 5.3 +/- 0.3 days after PGF2alpha treatment and the number of ovulated follicles was 15.5 +/- 1.4 detected at 7.6 +/- 0.2 days after the treatment. CONCLUSIONS: This study has defined the effective dose and timing of EDP treatment for inducing pseudopregnancy in cyclic pigs. Our results also indicated that EDP caused a lowering of inhibin concentrations during pseudopregnancy and small numbers of follicles from 20 to 28 days after EDP. In contrast, EDP-induced pseudopregnancy appears to have no adverse effect on follicular development and subsequent ovulation following PGF2alpha administration.

Heart fatty acid-binding protein offers similar diagnostic performance to high-sensitivity troponin T in emergency room patients presenting with chest pain.

BACKGROUND: The aim of the present study was to evaluate the diagnostic accuracy of high-sensitivity troponin T (hsTnT) in patients with suspected acute coronary syndrome (ACS) in comparison to heart fatty acid-binding protein (H-FABP), high-sensitivity C-reactive protein, myeloperoxidase (MPO), and pentraxin 3 (PTX3). METHODS AND RESULTS: Patients (n=432) with chest pain were recruited for the analysis. ACS was diagnosed in 298 patients (69%). The diagnostic accuracy of measurements obtained at presentation, as quantified by the area under the receiver operating curve (AUC), was highest for hsTnT (AUC=0.82; 95% confidence interval [CI]: 0.78-0.87) and H-FABP (AUC=0.83; 95%CI: 0.78-0.87). Sensitivity (87.9%) and negative likelihood (LH; 0.2) for hsTnT were the highest and lowest, respectively, but H-FABP had the highest specificity (78.5%) and positive LH (3.6). Among patients who presented within 2h after the onset of chest pain, MPO had the highest AUC (0.82; 95%CI: 0.69-0.94). Combined use of H-FABP and MPO measurements yielded a sensitivity of 69.2%, specificity of 84.2%, positive LH of 4.4, and negative LH of 0.4. CONCLUSIONS: The hsTnT assay offers excellent diagnostic performance to rule out ACS, but it is prone to false-positive results. H-FABP offers similar overall diagnostic performance, while the combination of H-FABP and MPO assays may improve the diagnosis of ACS, particularly in patients with recent onset of chest pain.

Peripheral concentrations of inhibin A, ovarian steroids, and gonadotropins associated with follicular development throughout the estrous cycle of the sow.

We investigated changes in peripheral concentrations of inhibin A, total inhibin, steroids, and gonadotropins throughout the intact estrous cycle of the sow in relation to ovarian changes determined by daily transrectal ultrasonography. All visible follicles of 3 mm or more in diameter were classified as small (> or =3 and <6 mm) or large (> or =6 mm). Follicular recruitment was identified in two periods of the cycle: one from the late luteal to the follicular phase, characterized by an increase in the number of small follicles followed by the appearance of large follicles; and another during the early luteal phase, consisting only of increased numbers of small follicles. Plasma concentrations of inhibin A increased (P<0.05), coinciding with the two periods of follicle emergence. Estradiol (E(2)) levels increased (P<0.05) during the follicular phase, but not during the early luteal phase. An inverse relationship (P<0.01) between the patterns of inhibin and FSH concentrations was noted around the two periods of follicle emergence, but there was no relationship (P> or =0.1) between the patterns of plasma E(2) and FSH during the early luteal phase. In conclusion, measurement of plasma inhibin A levels combined with ultrasonographic examination of the ovaries revealed two periods of synchronous follicular growth during the sow's estrous cycle. The results strongly suggest that inhibin A functions as a negative feedback regulator of FSH secretion throughout the estrous cycle, whereas E(2) appears to influence FSH secretion only during the follicular phase.

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.

Activation of extracellular signal-regulated kinase 5 reduces cardiac apoptosis and dysfunction via inhibition of a phosphodiesterase 3A/inducible cAMP early repressor feedback loop.

Substantial evidence suggests that the progressive loss of cardiomyocytes caused by apoptosis significantly contributes to the development of heart failure. beta-Adrenergic receptor activation and subsequent persistent phosphodiesterase 3A (PDE3A) downregulation and concomitant inducible cAMP early repressor (ICER) upregulation (PDE3A/ICER feedback loop) has been proposed to play a key role in the pathogenesis of cardiomyocyte apoptosis. In contrast, insulin-like growth factor-1 can activate cell survival pathways, providing protection against cell death and restoring muscle function. In this study, we found that insulin-like growth factor-1 activates extracellular signal-regulated kinase 5 (ERK5) and inhibits PDE3A/ICER feedback loop. Insulin-like growth factor-1 normalized isoproterenol-mediated PDE3A downregulation and ICER upregulation via ERK5/MEF2 activation, and also inhibited isoproterenol-induced myocyte apoptosis. To determine the physiological relevance of ERK5 activation in regulating PDE3A/ICER feedback loop, we investigated the PDE3A/ICER expression and cardiomyocyte apoptosis in transgenic mice with cardiac specific expression of a constitutively active form of mitogen-activated protein (MAP)/extracellular signal-regulated protein kinase (ERK) kinase 5alpha (MEK5alpha) (CA-MEK5alpha-Tg). In wild-type mice, pressure overload- or doxorubicin-induced significant reduction of PDE3A expression and subsequent ICER induction. Cardiac specific expression of CA-MEK5alpha rescued pressure overload- or doxorubicin-mediated PDE3A downregulation and ICER upregulation and inhibited myocyte apoptosis as well as subsequent cardiac dysfunction in vivo. These data suggest that preventing the feedback loop of PDE3A/ICER by ERK5 activation could inhibit progression of myocyte apoptosis as well as cardiac dysfunction. These data suggest a new therapeutic paradigm for end stage of heart failure by inhibiting the PDE3A/ICER feedback loop via activating ERK5.

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.

Role of p90 ribosomal S6 kinase-mediated prorenin-converting enzyme in ischemic and diabetic myocardium.

BACKGROUND: Epidemiological data strongly indicate that diabetes increases the incidence of heart failure. Although the benefit of angiotensin-converting enzyme inhibitor (ACE-I) treatment during and after myocardial infarction has been found to be greater in diabetics than nondiabetics and activation of the renin-angiotensin system (RAS) has been implicated, the molecular basis of these actions remains unclear. METHODS AND RESULTS: We generated transgenic mice with cardiac-specific overexpression of wild-type p90 ribosomal S6 kinase (WT-p90RSK-Tg) and a dominant-negative form of p90RSK (DN-p90RSK-Tg). Recovery of cardiac function after ischemia/reperfusion in WT-p90RSK-Tg isolated mouse hearts was significantly impaired. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry revealed specific induction of prorenin-converting enzyme (PRECE) in WT-p90RSK-Tg mice. mRNA induction of PRECE was confirmed with serial angiotensinogen protein reduction after perfusion in WT-p90RSK-Tg mice, suggesting an increase of angiotensinogen cleavage and subsequent RAS activation in WT-p90RSK-Tg mice. We investigated the role of the RAS in WT-p90RSK-Tg animals after ischemia/reperfusion with the use of an ACE-I (captopril) and an angiotensin II type 1 receptor blocker (olmesartan). We did not observe any effect of these inhibitors in non-Tg littermate controls, thus corroborating other reports in rodents. In contrast, both captopril and olmesartan significantly improved cardiac function and reduced infarct size in WT-p90RSK-Tg mice. At 8 months of age, WT-p90RSK-Tg mice developed cardiac dysfunction. p90RSK activity and PRECE mRNA were both increased by streptozotocin-induced hyperglycemia in non-Tg littermate controls, whereas DN-p90RSK-Tg animals exposed to streptozotocin did not have PRECE induction. CONCLUSIONS: This study demonstrates the critical role of p90RSK in hyperglycemia-mediated myocardial PRECE induction, which may explain the augmentation of the RAS in diabetic hearts and provide an alternative therapeutic approach to treat diabetic cardiomyopathy.

Inhibiting p90 ribosomal S6 kinase prevents (Na+)-H+ exchanger-mediated cardiac ischemia-reperfusion injury.

BACKGROUND: Pharmacological and genetic studies indicate that the (Na+)-H+ exchanger isoform 1 (NHE1) plays a critical role in myocardial ischemia and reperfusion (I/R) injury. We found that p90 ribosomal S6 kinase (RSK) phosphorylated serine 703 of NHE1, stimulating 14-3-3 binding and NHE1 activity. Therefore, we hypothesized that inhibiting RSK in cardiomyocytes would prevent NHE1 activation and decrease I/R-mediated injury. METHODS AND RESULTS: To examine the role of RSK in vivo, we generated transgenic mice with cardiac-specific overexpression of dominant negative RSK (DN-RSK-TG). DN-RSK-TG hearts demonstrated normal basal cardiac function and morphology. However, myocardial infarction (left coronary artery occlusion for 45 minutes) in DN-RSK-TG hearts was significantly reduced at 24 hours of reperfusion from 46.9+/-5.6% area at risk in nontransgenic littermate controls to 26.0+/-4.2% in DN-RSK-TG (P<0.01). Cardiomyocyte apoptosis was significantly reduced after I/R in DN-RSK (0.9+/-0.2%) compared with nontransgenic littermate controls (6.2+/-2.6%). Importantly, activation of RSK and interaction of 14-3-3 with NHE1, necessary for agonist-stimulated NHE1 activity, were increased by I/R and inhibited by 70% in DN-RSK-TG (P<0.01). Next, we transduced rat neonatal cardiomyocytes with adenovirus-expressing DN-RSK (Ad.DN-RSK) and measured NHE1 activity. The baseline rate of pH recovery in acid-loaded cells was equal in cells expressing LacZ or DN-RSK. However, NHE1 activation by 100 micromol/L H2O2 was significantly inhibited in cells expressing DN-RSK (0.16+/-0.02 pH units/min) compared with Ad.LacZ (0.49+/-0.13 pH units/min). Apoptosis induced by 12 hours of anoxia followed by 24 hours' reoxygenation was significantly reduced in cells expressing Ad.DN-RSK (18.6+/-2.0%) compared with Ad.LacZ (29.3+/-5.4%). CONCLUSIONS: In summary, RSK is a novel regulator of cardiac NHE1 activity by phosphorylating NHE1 serine 703 and a new pathological mediator of I/R injury in the heart.

Measurement of porcine luteinizing hormone concentration in blood by time-resolved fluoroimmunoassay.

We validated a time-resolved fluoroimmunoassay (TR-FIA) using the lanthanide element europium as a non-radiological tracer for measuring luteinizing hormone (LH) in porcine peripheral blood. The dose-response curve of the reference standard ranged from 0.2048 to 50 ng/ml. Good parallelism was noted between the LH standard and plasma sample. The profile of LH throughout the estrous cycle, assessed by daily blood sampling, was consistent with the previous findings obtained by radioimmunoassays (RIAs). Moreover, the secretory patterns of pulsatile LH in the follicular phase and the preovulatory LH surge were also similar to those obtained in previous RIAs. We conclude that TR-FIA can be used to measure LH levels in porcine blood, with practical and convenient applications.

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.

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.

The role of Notch signaling in diabetic endothelial progenitor cells dysfunction.

AIMS: To investigate the role of Notch signaling pathway in vasculogenic dysfunction of diabetic EPCs (DM-EPCs). METHODS: The study was performed in mice and diabetes was induced with Streptozotocin. The functional consequences of Notch pathway modulation were studied by assessment of colony forming capacity (EPC colony forming assay), EPC differentiation capacity (% of definitive EPC-CFU (dEPC-CFU)), circulating EPCs (EPC culture assay) and migrated cells (migration assay); in the presence of Notch inhibitor (γ-secretase inhibitors (GSI)) compared to control. Notch pathway and VEGF involvement in DM- EPCs were assessed by gene expression (RT-qPCR). RESULTS: DM demonstrated to increase Notch pathway expression in bone marrow (BM) EPCs followed by lower EPC-CFU number, EPCs differentiation capacity, number of circulating EPCs, migrated cells and VEGF expression compared to control (p<0.05). Inhibition of Notch pathway by GSI rescued vasculogenic dysfunction in DM-EPCs as represented by increase in EPC-CFU number, differentiation capacity and number of circulating EPCs (p<0.05). CONCLUSION: Our findings indicate the involvement of Notch pathway in mediating DM-EPCs dysfunction including less number of EPC-CFU, circulating EPCs and migrated cell number compared to control. Further in vitro inhibition of Notch pathway by GSI rescued DM-EPC dysfunction. Therefore targeting Notch pathway in DM may provide a target to restore DM-EPC dysfunction.

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.