Decrease In Annexin Research Articles

CD36 Promotes Platelet Phosphatidylserine Exposure through MAP Kinase ERK5

Dyslipidemic conditions can promote atherosclerotic plaque instability and rupture, leading to thrombosis that can cause life threatening complications. Thrombosis in this setting is augmented by circulating oxidized lipids on oxidized low density lipoprotein particles (oxLDL) that are recognized by key scavenger receptors. CD36 is a major platelet scavenger receptor that recognizes oxLDL and consequently promotes a prothrombotic platelet phenotype through multiple signaling pathways. We previously reported that recognition of oxLDL by CD36 generates specific reactive oxygen species through Src family kinases and NADPH oxidase. These species modulate redox sensitive signaling, such as the activation of the oxidant sensor MAP kinase ERK5, leading to platelet integrin activation, aggregation, and thrombus formation. Although ERK5 was a key effector for CD36 to promote thrombosis, the downstream signaling mechanisms are unclear. We hypothesize that CD36 promotes a procoagulant phenotype that requires ERK5. Platelets isolated and washed from healthy human donors were stimulated with buffer, native low-density lipoprotein (LDL), or oxLDL, and were stained with fluorophore-conjugated Annexin V or Lactadherin to detect exposure of procoagulant phosphatidylserine (PS). OxLDL-stimulated platelets showed dose-dependent 13.4 ± 2.8% and 18.5 ± 3.5% increases in Lactadherin binding at 50 and 100μg/mL oxLDL compared to control LDL (p=0.026; p<0.01), and a rapid binding within 5 min (11.0±0.35%, p<0.001). Furthermore, pre-treatment with oxLDL followed by addition of convulxin, a snake venom that activates platelets through the collagen signaling receptor GPVI, showed a 58.1 ± 4.62% increase in Lactadherin binding compared to oxLDL and convulxin alone (p<0.0001). Addition of ADP or thrombin, agonists that activates specific G-protein coupled receptors, following pre-treatment with oxLDL did not enhance Lactadherin binding. Platelets treated with FA6, a monoclonal CD36 antibody which prevents oxLDL binding, or BIX02188, an ERK5 inhibitor, showed an 80-90% decrease in Annexin V binding (p=0.039; p=0.02) after oxLDL exposure compared to a control non-immunizing IgG or DMSO. To link known pathways for PS exposure with oxLDL-induced PS exposure, platelets were stimulated with oxLDL followed by treatment with Z-VAD-FMK, a “pan” caspase inhibitor, or cyclosporin A, a cyclophilin D/mitochondrial permeability transition pore inhibitor. Z-VAD-FMK treated platelets showed 68% decrease in Lactadherin binding compared to control DMSO (p=0.0196), whereas cyclosporin A did not inhibit Lactadherin binding. Relevance of CD36/ERK5-mediated PS exposure was demonstrated using an in vivo arterial thrombosis model that relies on autologous transplantation of the epigastric artery, which has an adventitial surface that serves as a collagen-rich thrombogenic substrate for thrombosis, into the carotid artery. This model of thrombosis was chosen due to the potential impact of free radical generation in the standard ferric chloride or Rose Bengal-mediated arterial thrombosis models. Bone marrows from platelet ERK5-deficient mice (ERK5 flox/Pf4-cre+) or control mice (ERK5 flox/flox) were transplanted into irradiated atherogenic apoE null mice fed a high fat or control diet. Fibrin deposition, detected by a fluorophore-conjugated antibody to fibrin, was enhanced in hyperlipidemic conditions for mice with platelet ERK5. High fat diet-fed chimeric mice without ERK5 or animals on control diet do not show enhanced fibrin deposition. In conclusion, oxLDL promoted PS exposure through a CD36-Src family kinases-ERK5-caspase-dependent mechanism. PS exposure by this pathway potentially enhances fibrin deposition in an atherogenic arterial in vivo thrombosis model, which suggests a role for CD36 and ERK5 in thrombosis and coagulation in dyslipidemic conditions. DisclosuresNo relevant conflicts of interest to declare.

Alleviation of Memory Deficit by Bergenin via the Regulation of Reelin and Nrf-2/NF-κB Pathway in Transgenic Mouse Model.

The present study aims to determine the neuroprotective effect of Bergenin against spatial memory deficit associated with neurodegeneration. Preliminarily, the protective effect of Bergenin was observed against H2O2-induced oxidative stress in HT-22 and PC-12 cells. Further studies were performed in 5xFAD Tg mouse model by administering Bergenin (1, 30 and 60 mg/kg; orally), whereas Bergenin (60 mg/kg) significantly attenuated the memory deficit observed in the Y-maze and Morris water maze (MWM) test. Fourier transform-infrared (FT-IR) spectroscopy displayed restoration of lipids, proteins and their derivatives compared to the 5xFAD Tg mice group. The differential scanning calorimeter (DSC) suggested an absence of amyloid beta (Aβ) aggregation in Bergenin-treated mice. The immunohistochemistry (IHC) analysis suggested the neuroprotective effect of Bergenin by increasing Reelin signaling (Reelin/Dab-1) and attenuated Aβ (1–42) aggregation in hippocampal regions of mouse brains. Furthermore, IHC and western blot results suggested antioxidant (Keap-1/Nrf-2/HO-1), anti-inflammatory (TLR-4/NF-kB) and anti-apoptotic (Bcl-2/Bax/Caspase-3) effect of Bergenin. Moreover, a decrease in Annexin V/PI-stained hippocampal cells suggested its effect against neurodegeneration. The histopathological changes were reversed significantly by Bergenin. In addition, a remarkable increase in antioxidant level with suppression of pro-inflammatory cytokines, oxidative stress and nitric oxide production were observed in specific regions of the mouse brains.

Mechanistic insights into the protective effects of chlorogenic acid against indomethacin-induced gastric ulcer in rats: Modulation of the cross talk between autophagy and apoptosis signaling

BackgroundThis study aimed to investigate the gastroprotective effect of chlorogenic acid (CGA) against Indomethacin (IND)-induced gastric ulcer (GU) in rats and its underlying mechanism, especially through autophagic and apoptotic pathways. MethodsSeventy-five rats were divided into five groups; control, IND (50 mg/kg, p.o.), CGA (100 mg/kg, p.o., 14 days), IND pretreated with CGA (50 mg/kg or 100 mg/kg, p.o., 14 days). The stomach tissues were examined to calculate the ulcer index and analyze markers of autophagy (beclin-1, LC3-II/LC3-I and p62), lysosomal function (cathepsin-D) and apoptosis (Bcl-2, Bax and caspase-3), along with expression of Akt/mTOR pathway using western blot or ELISA techniques. In addition, viability of gastric mucosal cells was detected by flowcytometry. Structural changes were assessed histologically, while autophagic and apoptotic changes of gastric mucosa were observed by transmission electron microscopy. ResultsCGA exhibited a dose-dependent gastroprotective effect by reversing IND-induced accumulation of autophagic vacuoles, significant reduction in beclin-1, LC3-II/LC3-I, and p62 levels, and down-regulation of p-Akt/p-mTOR expression. CGA100 also restored normal autolysosomal function by modulation of cathepsin-D levels. Furthermore, pretreatment with CGA100 was significantly associated with an increase in antiapoptotic protein Bcl-2 along with a decrease in proapoptotic Bax and caspase-3 proteins in such a way that impairs IND-induced apoptosis. This was confirmed by CGA-induced significant decrease in annexin V+ cells. ConclusionsThe natural compound CGA offers a novel gastroprotective intervention against IND-induced GU through restoration of normal autophagic flux, impairment of apoptosis in a crosstalk mechanism mediated by Akt/mTOR pathway reactivation, and alleviation of IND-induced lysosomal dysfunction.

Change of annexin binding of monocytes as an expression of cellular response to Candida albicans: down-regulation in severe sepsis.

To study the differences of monocyte activation by albicans and non-albicans species of Candida and its change in sepsis, peripheral blood mononuclear cells were isolated from 17 healthy volunteers and 26 patients with severe sepsis/shock, and incubated in the absence/presence of heat-killed (HK) isolates of four different Candida species and purified β-D-glucan from C.albicans. Experiments were repeated in the presence and absence of inhibitors of intracellular activation pathways. Expression of annexin V on cells membranes of monocytes and lymphocytes, cytoplasmic activity of caspase-3, and DNA fragmentation of monocytes were studied. Membrane expression of annexin V on viable monocytes of healthy volunteers decreased significantly after incubation with C.albicans but not with non-albicans species. The decrease was dose-dependent from the Candida inoculum and by the concentration of β-D-glucan. A relationship with inhibition of apoptosis was found as the activity of caspase-3 activity, and the level of DNA fragmentation were also decreased. Incubation in the absence/presence of inhibitors showed that the decrease by annexin V expression resulted by activation of the dectin-1 pathway and Raf-1 by β-D glucan. The decrease of annexin V(+)/PI(-) expression was not shown on monocytes of patients with severe sepsis/shock, where no effect of inhibitors was found. Decrease of annexin V binding on monocytes can be viewed as a selective response to C.albicans partly effected through activation of dectin-1. This response is down-regulated after a septic insult.

Let-7b suppresses apoptosis and autophagy of human mesenchymal stem cells transplanted into ischemia/reperfusion injured heart 7by targeting caspase-3.

IntroductionMesenchymal stem cells (MSCs) have therapeutic potential for the repair of myocardial injury. The efficacy of MSC therapy for myocardial regeneration mainly depends on the survival of cells after transplantation into the infarcted heart. In the transplanted regions, reactive oxygen species (ROS) can cause cell death, and this process depends on caspase activation and autophagosome formation.MethodsA Software TargetScan was utilized to search for microRNAs (miRNAs) that target caspase-3 mRNA. Six candidate miRNAs including let-7b were selected and transfected into human MSCs in vitro. Expression of MEK-EKR signal pathways and autophagy-related genes were detected. Using ischemia/reperfusion model (I/R), the effect of MSCs enriched with let-7b was determined after transplantation into infarcted heart area. Miller catheter was used to evaluate cardiac function.ResultsHere, we report that let-7b targets caspase-3 to regulate apoptosis and autophagy in MSCs exposed to ROS. Let-7b-transfected MSCs (let-7b-MSCs) showed high expression of survival-related proteins, including p-MEK, p-ERK and Bcl-2, leading to a decrease in Annexin V/PI- and TUNEL-positive cells under ROS-rich conditions. Moreover, autophagy-related genes, including Atg5, Atg7, Atg12 and beclin-1, were significantly downregulated in let-7b-MSCs. Using a rat model of acute myocardial infarction, we found that intramyocardial injection of let-7b-MSCs markedly enhanced left ventricular (LV) function and microvessel density, in accordance with a reduced infarct size and the expression of caspase-3.ConclusionsTaken together, these data indicate that let-7b may protect MSCs implanted into infarcted myocardium from apoptosis and autophagy by directly targeting caspase-3 signaling.

Thymic Stromal Lymphopoeitin Signaling Contributes To Increased Risk Of Relapse Of Pediatric Acute Lympoblastic Leukemia Through Enhanced Survival Of Leukemic Cells That Overexpress Thymic Stromal Lymphopoeitin Receptor

Pediatric acute lymphoblastic leukemia (ALL) is the most common childhood malignancy. Although the cure rate for this disease is greater than 85%, ALL remains the number one cause of cancer-related deaths in children due to relapse of ALL. Therefore, there is a great need to identify new therapies for patients who have recurrent disease. Recently, a subset of pediatric ALL patients whose leukemic cells express high levels of thymic stromal lymphopoietin receptor (TSLPR/CRLF2) have been shown to have an increased risk of relapse and shorter disease free and poorer overall survival. Overexpression of TLSPR occurs in 8% of unscreened pediatric precursor B ALL and occurs by genomic rearrangement of the TSLPR gene, which fuses the unmutated TSLPR gene to altered transcriptional control or by other yet to be described means. The mechanism by which Thymic Stromal Lymphopoietin (TLSP) signaling contributes to increased risk of relapse is unknown. Studies have shown aberrant signaling in high TSLPR expressing ALL patient derived cell lines relying heavily on in vitro experiments. As no pre-clinical model of high TSLPR ALL has been published, we created a model of high TSLPR expressing leukemia to study TSLPR overexpressing leukemia progression. We have created a high TSLPR expressing leukemia cell line through retroviral transduction of a transplantable syngeneic mouse leukemia model in which the leukemic progression can be studied with physiologic levels of TSLP. This high TSLPR leukemia has levels of expression of TSLPR comparable to what is found on human leukemia that overexpress TSLPR. The TSLPR is functional in these cells and we see increased phosphorylation of STAT5 protein in response to IL-7 or TSLP stimulation. When we introduce the leukemia into mice and look at disease progression, we observed an 8 fold difference in the numbers of cells in the bone marrow 5 days after intravenous injection corresponding to an early stage of leukemia progression (Figure 1. high TSLPR 1.61%+/-0.95 vs. low TSLPR 0.20%+/-0.16). Interestingly we find no significant difference in long term survival of mice injected with either low or high TSLPR leukemia lines. The increased numbers of leukemic cells in the bone marrow at early stages of leukemic progression could be due to an increased rate of proliferation or better survival/engraftment. Low and high TSLPR expressing cells show no significant difference in growth rate in vitro or in vivo in dye dilution assays (Figure 2) suggesting that the increase in leukemic cells in the bone marrow is through enhanced survival. To test this, we treated low and high TSLPR leukemia lines with the steroid dexamethasone in the absence or presence of TSLP. We found that the addition of TSLP significantly reduced the Annexin V positive relative to cells not treated with TSLP in the high TSLP expressing leukemia cells, while in low TSLPR expressing cells we observed no decrease in Annexin V positive cells (Figure 3). This suggests that high TSLPR expression sensitizes leukemia cells to TSLP in the leukemia microenvironment. To confirm that this is the case we have found by gene expression analysis that we can detect TSLP in mouse bone marrow. We hypothesize that therapies targeting the TSLP signaling axis in ALL would decrease the risk of relapse. To test this hypothesis we have generated TSLPR-Fc conjugates to block TSLP signaling. We plan on using these reagents to block TSLP signaling to see if we can reverse the increased amounts of leukemia we find in mice at early stages of leukemic progression as well as the eliminate the survival advantage provided by TSLP to high TSLPR expressing leukemic cells in response to chemotherapeutic agents. [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

THU0041 Microparticle-Associated Poly(I:C) is Resistant to Rnase III Degradation and Protects Rheumatoid Arthritis Synovial Fibroblasts from Trail-Induced Apoptosis

BackgroundWe have shown recently that microparticles (MP) from monocytes stimulated with Poly(I:C) (PIC), a model for dsRNA, decrease the sensitivity of RA synovial fibroblasts (RASF) to TRAIL-induced apoptosis in an...

Differential expression of MMP‐2 and annexin V in normal and neoplastic mouse bone cells in response to direct expression of angiotensin II

Recent evidence has shown that Angiotensin II (Ang II) can stimulate growth in a number of different cell lines. We looked at the expression of MMP‐2, annexin V, and the Ang II receptor, AT1, to determine if these proteins are differentially regulated in normal (MC3T3) and cancerous (K7MZ) mouse bone cells. MMP‐2 has been shown to increase inflammation and metastasis, whereas annexin V has been linked to apoptosis. In order to assess whether the differences in MMP‐2 and annexin V expression were mediated by AT1, and to confirm that the Ang II produced in these cells acts via an intracrine pathway, we measured AT1 by Western blot analysis. In MC3T3 cells there was little change in MMP‐2 expression between control and the Ang II expressing cells. K7MZ mouse osteoblasts showed an increase in MMP‐2 expression as compared to the MC3T3 cells as well as an increase in expression between control and experimental (Ang II expressing) within the cell line. Cancer cells down regulate genes responsible for inducing apoptosis. Our data are consistent with this model as the K7MZ cells showed a marked decrease in annexin V expression in cells expressing angiotensin II directly. AT1 expression was not affected in either cell line by direct expression of Ang II. These data suggest that Ang II may play a role in the growth of cancer cells, and their ability to metastasize and avoid apoptosis.

Molecular Mechanisms of Pro-Survival and Differentiating Function of Bone Marrow-Derived Adipocytes On Acute Monoblastic Leukemia Cells.

AbstractAbstract 2582Survival of adult patients with acute monocytic leukemia remains dismal. We proposed the hypothesis that bone marrow (BM) adipocytes abundant in aging marrow microenvironment play a role in chemoresistance of AML cells. We have reported that BM adipocytes promote monocytic differentiation, activation, and survival of monoblastic leukemia cells (ASH, 2012).In this study, we examined the molecular mechanism of BM adipocytes causing survival and differentiation of monoblastic leukemia cells. Monoblastic leukemia cell line U937 and primary samples from acute monoblastic leukemia patients (n=6) were co-cultured with BM-derived marrow stromal cells (MSC) and MSC-differentiated adipocytes. Under limiting serum conditions, MSCs and adipocytes inhibited spontaneous cell death of U937 cells with decrease in subG1 fractions, more prominently in co-cultures with adipocytes than with MSCs (% subG1 fraction; cultured alone 23.9+9.0, co-cultured with MSC 15.7+1.9%, co-cultured with adipocytes 11.9+1.2%, p=0.04). Adipocytes but not MSC moderately reduced spontaneous apoptosis in primary leukemic blasts (n=6, p=0.02).We next focused on adipocytokines, lepin and plasminogen activator inhibitor 1(PAI-1), both implicated in cancer progression, as potential mediators of the protective effects by adipocytes. mRNA levels of leptin, leptin receptor and PAI-1 were up-regulated in MSC-derived adipocytes during adipocytic differentiation (relative expression to GAPDH, leptin: MSC 0, adipocyte 123.3+35.0; leptin receptor: MSC 0.16+0.05, adipocyte 4.47+0.37; PAI-1: MSC 314.9+46.5, adipocyte 3766.1+656.2). High level of secreted leptin was confirmed by ELISA in adipocyte conditioning medium (MSC 23.1+2.9, adipocyte 110.0+4.6 pg/mL). In serum-starved conditions, human recombinant leptin (5ng/mL) stimulated cell growth of monoblastic U937 cells known to express leptin receptor (35.8+7.9% increase of viable cell number at 48 hours, p=0.02) and inhibited apoptosis (%subG1 fraction; control 26.2+3.9, leptin16.4+4.1%, p=0.04).Human recombinant PAI-1 inhibited spontaneous and ara-C-induced apoptosis in U937 cells (11.2+1.1% and 15.1+1.7% decrease of Annexin V posivitity p=0.01). These results suggest that BM adipocytes produced leptin and PAI-1contribute to support myeloid leukemia cells survival within BM microenvironment.We next focused on molecular changes in U937 cells co-cultured with adipocytes. We found up-regulation of monocyte/macrophage differentiation gene PPARg and of PPARg target genes, CD36 and FABP4, by quantitative RT-PCR (relative expression to GAPDH, control vs co-culture with adipocytes: PPARg: 8.8+0.9 v.s.18.8+2.6, CD36; 105.0+11.8v.s.425.7+35.6; FABP4: 0.27+0.03v.s.153.7+12.8). The purity of U937 cells separated from adipocytes was confirmed by lack of CD90 mRNA expression by PCR. PPARγ is known to promote monocytic differentiation and uptake of oxidized cholesterol through the induction of scavenger receptor CD36, a protein marker of mature monocytes/macrophages. The up-regulation of cell surface CD36 expression by co-culture with adipocytes was indeed found in U937 cells and in 4 of the 6 primary AML samples by flow cytometory (increase in CD36 MFI: U937; 230.28, Pt samples; 46.4+20.2). These data suggest that BM adipocytes promote monocytic differentiation of monoblastic leukemia cells.In summary, our findings indicate that secretion of leptin and PAI-1 by BM adipocytes supports leukemic cell survival, while PAI-1 primarily and directly inhibits apoptosis of AML cells. It is conceivable that increased adipocyte content of BM in elderly AML patients may negatively affect the responsiveness of monocytic leukemia cells to chemotherapy. Disclosures:No relevant conflicts of interest to declare.

Modulation of Transcription Factor Nrf2 in an In Vitro Model of Traumatic Brain Injury

Traumatic brain injury (TBI) afflicts approximately 1.4 million people in the United States and TBIs have been labeled a major cause of death and disability on a global scale. Regulatory responses in a variety of neuronal loss conditions have supported the protective involvement of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) transcription factor. Nrf2 regulates antioxidant enzyme genes, and an increase in Nrf2 expression may counteract oxidative damage that results from TBI. Elevated Nrf2 may ultimately act through the upregulation of downstream target genes such as thioredoxin (Trx) and heat-shock protein-70 (HSP70) and this may reduce neuronal loss. We performed multiple mild biaxial stretch injuries to neuroblastoma cells in culture, and examined the effects of the Nrf2 activator, tert-butylhydroquinone (tBHQ). We also compared the stretch injury to oxidative insult. We confirmed that Trx and HSP70 were upregulated by treatment with tBHQ. We observed that tBHQ protected neurons from either insult, and that this was evident by different measures of cell viability and a decrease in annexin V binding. Neuronal health after insult was improved approximately 50% by tBHQ, indicating that neurons exposed to TBI in vitro can be protected.

Angiotensin II differentially regulates annexin V and glypican 6 in MC3T3 cells and B16 melanoma cells

Mouse MC3T3 cells (non‐cancerous) and B16 melanoma cells were transfected with a synthetic gene that expresses angiotensin II (Ang II) directly under the control of a CMV promoter. Previous studies using MALDI‐TOF/MS identified several candidate proteins for western blot analysis; Annexin V and Glypican 6 were chosen for further study. Annexin V is a member of a family of calcium‐binding proteins and has been implicated as a regulator of several processes including cell adhesion and apoptosis. One of the earliest events in apoptosis is the externalization of phosphatidylserine (PS) which is translocated from the cytoplasmic side of the plasma membrane to the cell surface soon after the induction of apoptosis, and that the Annexin V protein has a strong, specific affinity for PS. B16 Melanoma cells showed a marked decrease in Annexin V expression; whereas, MC3T3 cells showed a substantial increase in expression. Glypican 6 is a glycophosphotidylinositol anchored heparin sulfate proteoglycan involved in cell growth and division. It is a putative cell surface co‐receptor for growth factors, extracellular matrix proteins and proteases. Like Annexin V, Glypican 6 was down‐regulated in B16 cells and up‐regulated in MC3T3 cells. These results suggest that Ang II has specific growth effects which are differentially regulated in cancer and non‐cancer cells.

Oridonin induces apoptosis and senescence by increasing hydrogen peroxide and glutathione depletion in colorectal cancer cells

We recently demonstrated that oridonin could induce apoptosis and senescence of colon cancer cells in vitro and in vivo. However, the underlying mechanism remains unknown. In this study, the involvement of reactive oxygen species in oridonin-induced cell death and senescence was investigated in colon adenocarcinoma-derived SW1116 cells. Oridonin increased intracellular hydrogen peroxide levels and reduced the glutathione content in a dose-dependent manner. N-acetylcysteine, a reactive oxygen species scavenger, not only blocked the oridonin-induced increase in hydrogen peroxide and glutathione depletion, but also blocked apoptosis and senescence induced by oridonin, as evidenced by the decrease in Annexin V and senescence-associated β-galactosidase- positive cells and the inhibition of oridonin-induced upregulation of p53 and p16 and downregulation of c-Myc. Moreover, exogenous catalase could inhibit the increase in hydrogen peroxide and apoptosis induced by oridonin, but not the glutathione depletion and senescence. Furthermore, thioredoxin reductase (TrxR) activity was reduced by oridonin in vitro and in cells, which may cause the increase in hydrogen peroxide. In conclusion, the increase in hydrogen peroxide and glutathione depletion account for oridonin-induced apoptosis and senescence in colorectal cancer cells, and TrxR inhibition is involved in this process. Given the importance of TrxR as a novel cancer target in colon cancer, oridonin would be a promising clinical candidate. The mechanism of oridonin-induced inhibition of TrxR warrants further investigation.

Accumulation of the SET protein in HEK293T cells and mild oxidative stress: cell survival or death signaling

SET protein (I2PP2A) is an inhibitor of PP2A, which regulates the phosphorylated Akt (protein kinase B) levels. We assessed the effects of SET overexpression in HEK293T cells, both in the presence and the absence of mild oxidative stress induced by 50 μM tert-butyl hydroperoxide. Immunoblotting assays demonstrated that SET accumulated in HEK293T cells and increased the levels of phosphorylated Akt and PTEN; in addition, SET decreased glutathione antioxidant defense of cell and increased expression of genes encoding antioxidant defense proteins. Immunofluorescence analysis demonstrated that accumulated SET was equally distributed in cytoplasm and nucleus; however, in cells that had been exposed to oxidative stress, SET was found in large aggregates in the cytoplasm. SET accumulation in HEK293T cells correlated with inhibition of basal apoptosis as evidenced by a decrease in annexin V staining and activity of caspases; under mild oxidative stress, SET accumulation correlated with caspase-independent cell death, as evidenced by increased PI and annexin V/PI double staining. The results suggest that accumulated SET could act via Akt/PTEN either as cell survival signal or as oxidative stress sensor for cell death.

The MAPK ERK1 Is a Sensor of the Steady-State Splenic Erythropoiesis.

Abstract 73In different culture models, conflicting results have been obtained with respect to the role of the ERK/MAPK pathway and the ERK kinases on erythropoiesis. There is no in vivo experimental data on the role of these kinases in adult erythropoeisis. The existence of two ERK isoforms (ERK1 and ERK2) suggests that they could play specific role, based on their expression, their activation level and/or the ratio between both of them. The ERK1−/− mice were used to study this hypothesis. Increased number of circulating erythrocytes, increased hemoglobin level and hematocrit were found in these mice. The deletion of ERK1 leads to an uncontrolled splenic erythropoiesis while the bone marrow erythropoiesis remains normal. The ERK1−/− mice display splenomegaly characterized by a marked expansion of the red pulp and an increased number in basophilic (Ery.A) and late basophilic (Ery.B) erythroblasts. This impaired erythropoiesis in ERK1−/− mice is cell autonomous as shown by bone marrow transplantation experiments. This splenic erythropoiesis is not due to an overexpression or overactivation of the ERK2 isoform in erythroblasts. It has been shown that Fas-mediated apoptosis of erythroblasts would limit the basal erythropoietic rate. In ERK1−/− mice, Ery.A expansion is associated with a decrease in cell surface expression of both Fas and FasL as compared with wild-type mice. This fall in Fas/FasL expression is correlated with a decrease in Annexin V binding on splenic Ery.A and Ery.B. In addition, cell cycle analysis revealed an increased S-phase in ERK1−/− Ery.A cells compared with wild-type Ery.A. In conclusion, these data demonstrate for the first time the in vivo involvement of the ERK/MAPK pathway in adult splenic erythropoiesis and underlies the specific role of ERK1 in this function. By regulating the cell surface expression of Fas and FasL on splenic erythroblasts, ERK1 acts as a sensor of the basal erythropoietic rate. Disclosures:No relevant conflicts of interest to declare.

A novel dynamic matrix detachment model reveals a shift from apoptosis to necrosis in melanoma cells

Anchorage-independence is a hallmark of invasive cancer. The setback of the classical poly-HEMA static matrix detachment (SMD) anoikis model is the absence of dynamic fluid circulation, resulting in cell aggregates. We addressed this problem by developing a novel 3D cell culture dynamic matrix detachment (DMD) model with a turbulent-free laminar flow, yielding a very low shear stress. In this study, we focused on melanoma cells where apoptosis was evaluated both via annexin V flow cytometry and caspase cleavage. The DMD model was superior to SMD in the induction of melanoma cell death and in revealing a shift from apoptosis to necrotic cell death, as evident by failure to activate caspase 9 and a decrease in annexin V stain. Combination of DMD with cisplatin could further accentuate necrotic cell death in cisplatin-resistant melanoma cells. Thus, the DMD model may be a useful matrix deprivation model to identify necrotic vs. apoptotic cell death pathways.

Neuroprotective effects of neuregulin-1 on B35 neuronal cells following ischemia

We previously showed that neuregulin-1 (NRG-1) protected neurons from death in vivo following focal ischemia. The goal of this study was to develop an in vitro rat ischemia model to examine the cellular and molecular mechanisms involved in the neuroprotective effects of NRG-1 on ischemia-induced neuronal death. Rat B-35 neuroblastoma cells differentiated by serum withdrawal, developed enhanced neuronal characteristics including, neurite extension and upregulation of neuronal markers of differentiation. When B35 neurons were subjected to oxygen glucose deprivation (OGD)/reoxygenation or glutamate, widespread neuronal death was seen after both treatments. Treatment with NRG-1 immediately after OGD significantly increased neuronal survival. NRG-1 administration also resulted in a significant decrease in annexin V, an early marker of apoptosis. However, the neurotoxic actions of glutamate were unaffected by NRG-1. The neuroprotective effects of NRG-1 were prevented by an inhibitor of the phosphatidylinositol-3-kinase/Akt pathway. These results provide a new model to gain insight into the mechanisms employed by NRG-1 to protect neurons from ischemic brain injury.

Dipyridamole Decreases Protease-Activated Receptor and Annexin-V Binding on Platelets of Poststroke Patients with Aspirin Nonresponsiveness

Background: Although controversial, the phenomenon of aspirin resistance (AR) has been correlated in some small studies with poor clinical outcomes in patients with coronary artery disease. Even less is known regarding the role of AR in the poststroke population. The reason for and the underlying mechanism of AR is unknown. We hypothesized that excessive formation of thrombin on the platelet surface may contribute to this phenomenon and assessed how dipyridamole affects multiple platelet and thrombin generation biomarkers in AR patients after ischemic stroke. Methods: Whole blood samples from 20 poststroke AR patients were pretreated with dipyridamole, simulating the therapeutic range, and then incubated for 45 min at 37°C. Platelet characteristics were assessed by aggregometry, cartridge-based analyzer, and receptor expression by flow cytometry. Markers of thrombin generation were measured in the autologous plasma by ELISA. Results: Pretreatment of blood with dipyridamole resulted in 22–26% diminished expression of intact PAR-1 receptor (p = 0.021 and p = 0.024) and 28–31% decrease of annexin V binding (p = 0.031 and p = 0.02) after incubation with 2 µg/ml and 4 µg/ml of dipyridamole, respectively. Platelet aggregation and thrombin generation markers were not affected in vitro by dipyridamole. Conclusions: Dipyridamole may be capable of overcoming increased prothrombinase complex formation and be in part able to compensate for AR in patients with moderate carotid stenosis. This phenomenon may explain the clinical advantages of Aggrenox^®, known to reduce ischemic events in poststroke patients as proven in clinical trials, though an additional antithrombotic benefit beyond the platelet inhibition by aspirin alone.

Akt Activation Regulates Macrophage Survival and Differentiation: Role of M-CSF and Endogenous ROS.

Previous reports from our laboratory showed M-CSF promotes PI 3-kinase activation resulting in the production of reactive oxygen species (ROS) and PI 3-kinase inhibitors, and the antioxidants diphenyleneiodonium (DPI) and n-acetyl cysteine (NAC) suppressed M-CSF-stimulated Erk activation. In this study, we hypothesized that M-CSF-induced generation of ROS affected Akt1 activation and sought to define the role of Akt1 in monocyte/macrophage survival and differentiation. We found that the production of ROS following M-CSF-treatment was inhibited by the antioxidant DPI. The addition of either DPI or NAC to the monocytes in the presence of M-CSF resulted in decrease cell survival as measured by Annexin V/PI and DNA fragmentation. In the cells treated with the antioxidants, there was a reduction in pAKT protein levels compared to M-CSF alone treatment suggesting that ROS contributed to Akt activity and cell survival. Macrophages from p47phox −/− mice, lacking a key component of the NADPH oxidase complex required for ROS generation were examined for M-CSF-induced survival and Akt1 activation. Bone marrow macrophages from p47phox −/ − mice and wild type (WT) littermates were isolated and differentiated in RPMI-1640 medium in the presence of 20 ng/ml of M-CSF for 5 days. We observed a reduction in Akt1 phosphorylation, cellular survival and increase in apoptosis measured by Annexin V/PI staining in p47phox −/ − macrophages compared to WT controls.Since macrophages from the p47phox−/− mice had reduced Akt1 activity and cell survival to M-CSF stimulation, we next wanted to independently evaluate the role of Akt in macrophage survival and differentiation. We therefore examined macrophages from mice that had targeted expression of activated Akt1 (Myr-Akt1) in mononuclear phagocytes. Bone marrow from Myr-Akt1 mice and (WT) littermates was isolated and cultured in the presence of 20 ng/ml of M-CSF for 5 days. We found that Myr-Akt1 cells had enhanced survival and reduced apoptosis versus WT cells. Interestingly, Myr-Akt1 mice had normal circulating numbers of monocytes, but had splenomegaly and increased numbers of mature macrophages in their spleens by CD68 staining. Since the expression of Myr-Akt1 in BMM enhanced cell survival, we were interested in the effect in human monocytes. Peripheral blood monocytes (PBM) were transiently transfected with Myr-Akt1 constructs. We observed a decrease in Annexin V/PI staining indicating promotion of cell survival in PBM expressing Myr-Akt compared to untransfected PBM. Since Akt1 appeared to be important for macrophage survival/differentiation, we examined its contribution in human alveolar macrophage homeostasis and found that freshly isolated macrophages had constitutive Akt1 activity and had increased levels of Akt1 protein. This data support a critical role for Akt1 in macrophage differentiation in mice. Together, our findings may provide insight in the pathogenesis of monocyte/macrophage homeostasis.

Annexin 2 Promotes the Formation of Lipid Microdomains Required for Calcium-regulated Exocytosis of Dense-Core Vesicles

Annexin 2 is a calcium-dependent phospholipid-binding protein that has been implicated in a number of membrane-related events, including regulated exocytosis. In chromaffin cells, we previously reported that catecholamine secretion requires the translocation and formation of the annexin 2 tetramer near the exocytotic sites. Here, to obtain direct evidence for a role of annexin 2 in exocytosis, we modified its expression level in chromaffin cells by using the Semliki Forest virus expression system. Using a real-time assay for individual cells, we found that the reduction of cytosolic annexin 2, and the consequent decrease of annexin 2 tetramer at the cell periphery, strongly inhibited exocytosis, most likely at an early stage before membrane fusion. Secretion also was severely impaired in cells expressing a chimera that sequestered annexin 2 into cytosolic aggregates. Moreover, we demonstrate that secretagogue-evoked stimulation triggers the formation of lipid rafts in the plasma membrane, essential for exocytosis, and which can be attributed to the annexin 2 tetramer. We propose that annexin 2 acts as a calcium-dependent promoter of lipid microdomains required for structural and spatial organization of the exocytotic machinery.

Lymphocyte’s Activation and Apoptosis After Coronary Artery Bypass Graft: A Comparative Study of Two Membrane Oxygenators–One with and Another without a Venous-Arterial Shunt

Newer oxygenators with the latest technologies are designed to attenuate the immune response, including lymphopenia, prompted by cardiopulmonary bypass (CPB) in cardiac surgery. We evaluated the effect of CPB, comparing an oxygenator with a venous-arterial shunt and a conventional oxygenator with regard to lymphocyte's early activation and apoptosis induction and its implications in post-CPB lymphopenia. Patients undergoing coronary artery bypass graft surgery with CPB, using either a conventional oxygenator or one with a venous-arterial shunt, had blood samples drawn at anesthetic induction (baseline); the beginning and end of the CPB; and at 6, 12, and 24 hours after surgery. Analysis by flow cytometry was undertaken to assess the expression of lymphocyte surface markers (CD3+, CD25+, CD26+, CD69+) and apoptosis (annexin V). Twenty patients were studied; 10 used a conventional oxygenator, and 10 used an oxygenator with venous-arterial shunt. Postoperative lymphopenia (50% decrease), 35% increased expression of CD69+, and 56% decrease in annexin V were significant comparing baseline to 24 hour value, similarly in both groups. Early activation (expression of CD69+) and degree of apoptosis (expression of annexin V) of lymphocytes after CBP in cardiac surgery was similarly observed in both types of oxygenators. The observed lymphopenia after CPB does not appear to be secondary to apoptosis.