Regression/eradication of gliomas in mice by a systemically-deliverable ATF5 dominant-negative peptide.

Malignant gliomas have poor prognosis and urgently require new therapies. Activating Transcription Factor 5 (ATF5) is highly expressed in gliomas, and interference with its expression/function precipitates targeted glioma cell apoptosis in vitro and in vivo. We designed a novel deliverable truncated-dominant-negative (d/n) form of ATF5 fused to a cell-penetrating domain (Pen-d/n-ATF5-RP) that can be intraperitoneally/subcutaneously administered to mice harboring malignant gliomas generated; (1) by PDGF-B/sh-p53 retroviral transformation of endogenous neural progenitor cells; and (2) by human U87-MG xenografts. In vitro Pen-d/n-ATF5-RP entered into glioma cells and triggered massive apoptosis. In vivo, subcutaneously-administered Pen-d/n-ATF5-RP passed the blood brain barrier, entered normal brain and tumor cells, and then caused rapid selective tumor cell death. MRI verified elimination of retrovirus-induced gliomas within 8-21 days. Histopathology revealed growth-suppression of intracerebral human U87-MG cells xenografts. For endogenous PDGF-B gliomas, there was no recurrence or mortality at 6-12 months versus 66% mortality in controls at 6 months. Necropsy and liver-kidney blood enzyme analysis revealed no adverse effects on brain or other tissues. Our findings thus identify Pen-d/n-ATF5-RP as a potential therapy for malignant gliomas.

TGRL Lipolysis Products Induce Stress Protein ATF3 via the TGF-ß Receptor Pathway in Human Aortic Endothelial Cells.

Studies have suggested a link between the transforming growth factor beta 1 (TGF-ß1) signaling cascade and the stress-inducible activating transcription factor 3 (ATF3). We have demonstrated that triglyceride-rich lipoproteins (TGRL) lipolysis products activate MAP kinase stress associated JNK/c-Jun pathways resulting in up-regulation of ATF3, pro-inflammatory genes and induction of apoptosis in human aortic endothelial cells. Here we demonstrate increased release of active TGF-ß at 15 min, phosphorylation of Smad2 and translocation of co-Smad4 from cytosol to nucleus after a 1.5 h treatment with lipolysis products. Activation and translocation of Smad2 and 4 was blocked by addition of SB431542 (10 µM), a specific inhibitor of TGF-ß-activin receptor ALKs 4, 5, 7. Both ALK receptor inhibition and anti TGF-ß1 antibody prevented lipolysis product induced up-regulation of ATF3 mRNA and protein. ALK inhibition prevented lipolysis product-induced nuclear accumulation of ATF3. ALKs 4, 5, 7 inhibition also prevented phosphorylation of c-Jun and TGRL lipolysis product-induced p53 and caspase-3 protein expression. These findings demonstrate that TGRL lipolysis products cause release of active TGF-ß and lipolysis product-induced apoptosis is dependent on TGF-ß signaling. Furthermore, signaling through the stress associated JNK/c-Jun pathway is dependent on TGF-ß signaling suggesting that TGF-ß signaling is necessary for nuclear accumulation of the ATF3/cJun transcription complex and induction of pro-inflammatory responses.

TCDD and omeprazole prime platelets through the aryl hydrocarbon receptor (AhR) non-genomic pathway.

The role of the aryl hydrocarbon receptor (AhR) in hemostasis has recently gained increased attention. Here, we demonstrate, by qRT-PCR and western blot, that human platelets express both AhR mRNA and AhR protein. AhR protein levels increase in a dose dependent manner when incubated with either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or omeprazole. Treatment of platelets with puromycin blocks increased AhR protein synthesis in the presence of AhR activators. Additionally, treatment of platelets with either activator results in phosphorylation of p38MAPK and cPLA2, two key signaling molecules in platelet activation pathways. Using the AhR competitive inhibitors alpha naphthoflavone and CH-223191, we show that phosphorylation of p38MAPK is AhR dependent. Further, inhibition of p38MAPK blocks downstream cPLA2 phosphorylation induced by TCDD or omeprazole. Treatment with AhR activators results in platelet priming, as demonstrated by increased platelet aggregation, which is inhibited by AhR antagonists. Our data support a model of the platelet AhR non-genomic pathway in which treatment with AhR activators results in increased expression of the AhR, phosphorylation of p38MAPK and cPLA2, leading to platelet priming in response to agonist.

Induction of ATF3 gene network by triglyceride-rich lipoprotein lipolysis products increases vascular apoptosis and inflammation.

OBJECTIVE: Elevation of triglyceride-rich lipoproteins (TGRLs) contributes to the risk of atherosclerotic cardiovascular disease. Our work has shown that TGRL lipolysis products in high physiological to pathophysiological concentrations cause endothelial cell injury; however, the mechanisms remain to be delineated. APPROACH AND RESULTS: We analyzed the transcriptional signaling networks in arterial endothelial cells exposed to TGRL lipolysis products. When human aortic endothelial cells in culture were exposed to TGRL lipolysis products, activating transcription factor 3 (ATF3) was identified as a principal response gene. Induction of ATF3 mRNA and protein was confirmed by quantitative reverse-transcription polymerase chain reaction and Western blot respectively. Immunofluorescence analysis showed that ATF3 accumulated in the nuclei of cells treated with lipolysis products. Nuclear expression of phosphorylated c-Jun N-terminal kinase (JNK), previously shown to be an initiator of the ATF3 signaling cascade, also was demonstrated. Small interfering RNA (siRNA)-mediated inhibition of ATF3 blocked lipolysis products-induced transcription of E-selectin and interleukin-8, but not interleukin-6 or nuclear factor-κB. c-Jun, a downstream protein in the JNK pathway, was phosphorylated, whereas expression of nuclear factor-κB-dependent JunB was downregulated. Additionally, JNK siRNA suppressed ATF3 and p-c-Jun protein expression, suggesting that JNK is upstream of the ATF3 signaling pathway. In vivo studies demonstrated that infusion of TGRL lipolysis products into wild-type mice induced nuclear ATF3 accumulation in carotid artery endothelium. ATF3(-/-) mice were resistant to vascular apoptosis precipitated by treatment with TGRL lipolysis products. Also peripheral blood monocytes isolated from postprandial humans had increased ATF3 expression as compared with fasting monocytes. CONCLUSIONS: This study demonstrates that TGRL lipolysis products activate ATF3-JNK transcription factor networks and induce endothelial cells inflammatory response.

Comparative Analysis of the Immunomodulatory Properties of Equine Adult-Derived Mesenchymal Stem Cells().

Mesenchymal stem cells (MSCs) derived from bone marrow (BM), adipose tissue (AT), umbilical cord blood (CB), and umbilical cord tissue (CT) are increasingly being used to treat equine inflammatory and degenerative lesions. MSCs modulate the immune system in part through mediator secretion. Animal species and MSC tissue of origin are both important determinants of MSC function. In spite of widespread clinical use, how equine MSCs function to heal tissues is fully unknown. In this study, MSCs derived from BM, AT, CB, and CT were compared for their ability to inhibit lymphocyte proliferation and secrete mediators in response to activation. Five MSC lines from each tissue were isolated. Lymphocyte proliferation was assessed in a mixed leukocyte reaction, and mediator secretion was determined by ELISA. Regardless of tissue of origin, quiescent MSCs did not alter lymphocyte proliferation or secrete mediators, except for transforming growth factor-ß (TGF-ß1). When stimulated, MSCs of all tissue types decreased lymphocyte proliferation, increased prostaglandin (PGE(2)) and interleukin-6 (IL-6) secretion, and decreased production of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). BM-MSCs and CB-MSCs also produced nitric oxide (NO), while AT-MSCs and CT-MSCs did not. Equine MSCs did not produce indoleamine 2,3-dioxygenase (IDO). These data suggest that activated equine MSCs derived from BM, AT, CT, and CB secrete high concentration of mediators and are similar to MSCs from rodents and humans in their immunomodulatory profiles. These findings have implication for the treatment of inflammatory lesions dominated by activated lymphocytes and TNF-α and IFN-γ in vivo.

Seasonal influences on CAPs exposures: differential responses in platelet activation, serum cytokines and xenobiotic gene expression.

Increasing evidence suggests a role for a systemic pro-coagulant state in the pathogenesis of cardiac dysfunction subsequent to inhalation of airborne particulate matter (PM). We evaluated platelet activation, systemic cytokines and pulmonary gene expression in mice exposed to concentrated ambient particulate matter (CAPs) in the summer of 2008 (S08) and winter of 2009 (W09) from the San Joaquin Valley of California, a region with severe PM pollution episodes. Additionally, we characterized the PM from both exposures including organic compounds, metals, and polycyclic aromatic hydrocarbons. Mice were exposed to an average of 39.01 µg/m(3) of CAPs in the winter and 21.7 µg/m3 CAPs in the summer, in a size range less than 2.5 µm for 6 h/day for 5 days per week for 2 weeks. Platelets were analyzed by flow cytometry for relative size, shape, CD41, P-selectin and lysosomal associated membrane protein-1 (LAMP-1) expression. Platelets from W09 CAPs-exposed animals had a greater response to thrombin stimulation than platelets from S08 CAPs-exposed animals. Serum cytokines were analyzed by bead based immunologic assays. W09 CAPs-exposed mice had elevations in IL-2, MIP-1α, and TNFα. Laser capture microdissection (LCM) of pulmonary vasculature, parenchyma and airways all showed increases in CYP1a1 gene expression. Pulmonary vasculature showed increased expression of ICAM-1 and Nox-2. Our findings demonstrate that W09 CAPs exposure generated a greater systemic pro-inflammatory and pro-coagulant response to inhalation of environmentally derived fine and ultrafine PM. Changes in platelet responsiveness to agonists, seen in both exposures, strongly suggests a role for platelet activation in the cardiovascular and respiratory effects of particulate air pollution.

Comparative gene responses to collected ambient particles in vitro: endothelial responses.

Epidemiologic studies associate exposure to ambient particulate matter (APM) with increased cardiovascular mortality. Since both pulmonary inflammation and systemic circulation of ultrafine particles are hypothesized as initiating cardiovascular effects, we examined responses of potential target cells in vitro. Human aortic endothelial cells (HAEC) were exposed to 10 µg/ml fine and ultrafine APM collected in an urban setting in summer 2006 or winter 2007 in the San Joaquin Valley, California. RNA isolated after 3 h was analyzed with high-density oligonucleotide arrays. Summer APM treatment affected genes involved in xenobiotic and oxidoreductase activity, transcription factors, and inflammatory responses in HAEC, while winter APM had a robust xenobiotic but lesser inflammatory response. Real-time polymerase chain reaction analysis confirmed that particulate matter (PM)-treated HAEC increased mRNA levels of xenobiotic response enzymes CYP1A1, ALDH1A3, and TIPARP and cellular stress response transcription factor ATF3. Inflammatory response genes included E-selectin, PTGS2, CXCL-2 (MIP-2α), and CCL-2 (MCP-1). Multiplex protein assays showed secretion of IL-6 and MCP-1 by HAEC. Since induction of CYP1A1 is mediated through the ligand-activated aryl hydrocarbon receptor (AhR), we demonstrated APM induced AhR nuclear translocation by immunofluorescence and Western blotting and activation of the AhR response element using a luciferase reporter construct. Inhibitor studies suggest differential influences of polycyclic aromatic hydrocarbon signaling, ROS-mediated responses and endotoxin alter stress and proinflammatory endothelial cell responses. Our findings demonstrate gene responses correlated with current concepts that systemic inflammation drives cardiovascular effects of particulate air pollution. We also demonstrate a unique pattern of gene responses related to xenobiotic metabolism in PM-exposed HAEC.

Differential cellular responses to protein adducts of naphthoquinone and monocrotaline pyrrole.

Protein-xenobiotic adducts are byproducts of xenobiotic metabolism. While there is a correlation between protein adduction and target organ toxicity, a cause and effect relationship is not often clear. Naphthoquinone (NQ) and monocrotaline pyrrole (MCTP) are two pneumotoxic electrophiles that form covalent adducts with a similar select group of proteins rich in reactive thiols. In this study, we treated human pulmonary artery endothelial cells (HPAEC) with NQ, MCTP, or preformed NQ or MCTP adducts to the protein galectin-1 (gal-1) and examined indicators of reactive oxygen species (ROS) oxidative injury, markers of apoptosis (caspase-3 and annexin V), and gene responses of cellular stress. ROS production was assayed fluorescently using CM-H(2)DCFDA. NQ adducts to gal-1 (NQ-gal) produced 183% more intracellular ROS than gal-1 alone (p < 0.0001). Caspase-3 activity and annexin V staining of phosphatidylserine were used to assess apoptotic activity in treated cells. HPAEC exposed to MCTP-gal had increases in both caspase-3 activation and membrane translocation of annexin V relative to gal-1 alone (p < 0.0001). Direct application of NQ produced significantly more ROS and induced significant caspase-3 activation, whereas MCTP did not. Human bronchial epithelial cells were also exposed to MCTP-gal and found to have significant increases in both caspase-3 activation and annexin V staining in comparison to that of gal-1 (p < 0.05). Western blot analysis showed that both NQ and MCTP significantly induced the Nrf2 mediated stress response pathway despite differences in ROS generation. ER stress was not induced by either adducts or parent compounds as seen by quantitative RT-PCR, but HOX-1 expression was significantly induced by NQ-gal and MCTP alone. Electrophile adduction to gal-1 produces different cytotoxic effects specific to each reactive intermediate.

Overexpression of CD133 promotes drug resistance in C6 glioma cells.

Glioblastoma multiforme is an extremely aggressive and clinically unresponsive form of cancer. Transformed neoplastic neural stem cells, resistant to chemotherapy and radiation therapy, are thought to be responsible for the initial tumor formation and the recurrence of disease following surgical resection. These stem cells express multidrug resistance markers along with CD133. We show that ectopic overexpression of CD133 in rat C6 glioma cells leads to significant reluctance to undergo apoptosis from camptothecin and doxorubicin. Although p53 was upregulated in CD133-overexpressing glioma cells treated with DNA-damaging agents, apoptosis seems to be p53 independent. At least one ABC transporter, rat P-glycoprotein/ABCB1, was upregulated by 62% in CD133(+) cells with a corresponding increase in activity. Thus, the combination of higher P-glycoprotein mRNA transcription and elevated transporter activity seems to contribute to the protection from cytotoxic reagents. In conclusion, previous investigators have reported that resilient cancer stem cells coexpress CD133 and ABC transporters with increased reluctance toward apoptosis. Our data suggest that CD133 may contribute to the observed resistance to apoptosis of CD133(+) cancer stem cells.

Smad signaling in the rat model of monocrotaline pulmonary hypertension.

Mutations in the bone morphogenetic protein receptor type II (BMPrII) gene have been implicated in the development of familial pulmonary artery hypertension (PAH). The function of BMP signal transduction within the pulmonary vasculature and the role BMPrII mutations have in the development of PAH are incompletely understood. We used the monocrotaline (MCT) model of PAH to examine alterations in Smad signal transduction pathways in vivo. Lungs harvested from Sprague-Dawley rats treated with a single 60-mg/kg intraperitoneal (IP) injection of MCT were compared to saline-treated controls 2 weeks following treatment. Smad 4 was localized by immunohistochemistry to endothelial nuclei of the intra-acinar vessels undergoing remodeling. Smad 4, common to both BMP and transforming growth factor beta (TGFbeta) signaling, and BMP-specific Smad 1 were significantly decreased in western blot from whole lungs of treated animals, while no change was found for TGFbeta-specific Smad 2. MCT-treated rats also had increased expression of phosphorylated Smad 1 (P-Smad 1) but not phosphorylated Smad 2 (P-Smad 2). There was a decrease in the expression of the full BMPrII protein but not its short form variant in MCT-treated rat lungs. The type I receptor Alk1 had increased expression. Collectively, our data indicate that vascular remodeling in the MCT model is associated with alterations in BMP receptors and persistent endothelial Smad 1 signaling.

Differences in taurine synthesis rate among dogs relate to differences in their maintenance energy requirement.

Diet-induced (taurine deficiency) dilated cardiomyopathy is reported more in large than small dogs possibly because taurine biosynthesis rate (TBR) is lower in large than small dogs. The TBR in 6 mongrels (37.9 +/- 2.1 kg) and 6 beagles (12.8 +/- 0.4 kg) was determined from the fractional dilution rate of urinary [1,2-(2)H(2)]-taurine, (d4-tau). All dogs were given a 15.6% protein, 0.60% sulfur amino acid (SAA) diet in amounts to maintain an ideal body condition score. After 3 mo, 14.6 mg/kg body weight of d4-tau was given orally and TBR determined from d4-tau to taurine ratio in urine collected each d for 6 d. Enrichments of d4-tau were determined by GC-MS. Thereafter, mongrels and beagles were paired by ranking of SAA intake per metabolic body weight per kg(0.75). Each pair received the same amount of diet/kg(0.75) for 2 wk, then TBR was again determined. Concentrations of taurine in plasma, blood, and urine and concentrations of plasma thiols were measured during each TBR determination. In Expt. 1, TBR and taurine concentrations in plasma and urine of mongrels were lower (P < 0.05) than those of beagles. In Expt. 2, TBR and taurine concentrations in blood and plasma of mongrels were lower (P < 0.05) than beagles. Together, the results support the hypothesis that large compared with small dogs have lower TBR when fed diets near-limiting in dietary SAA, but adequate to maintain ideal body condition.

Lipolysis products from triglyceride-rich lipoproteins increase endothelial permeability, perturb zonula occludens-1 and F-actin, and induce apoptosis.

Products generated from lipoprotein lipase-mediated hydrolysis of triglyceride-rich lipoproteins (TGRL) are reported to increase endothelial layer permeability. We hypothesize that these increases in permeability result from the active rearrangement and dissolution of the junctional barrier in human aortic endothelial cells, as well as induction of the apoptotic cascade. Human aortic endothelial cells were treated with TGRL lipolysis products generated from coincubation of human TGRL plus lipoprotein lipase. Measurement of transendothelial electrical resistance demonstrated a time-dependent decrease in endothelial barrier function in response to TGRL lipolysis products. Immunofluorescent localization of zonula occludens-1 (ZO-1) showed radial rearrangement along cell borders after 1.5 h of treatment with lipolysis products. A concurrent redistribution of F-actin from the cell body to the cell margins was observed via rhodamine phalloidin staining. Immunofluorescent imaging for occludin and vascular endothelial cadherin showed that these proteins relocalize as well, although these changes are less prominent than for ZO-1. Western analysis of cells exposed to lipolysis products for 3 h revealed the fragmentation of ZO-1, a reduction in occludin, and no change of vascular endothelial cadherin. Lipolysis products also increased caspase-3 activity and induced nuclear fragmentation. Treatments did not cause oncosis in cells at any point during the incubation. These results demonstrate that TGRL lipolysis products play an important role in the regulation of endothelial permeability, the organization of the actin cytoskeleton, the localization and expression of junctional proteins, especially ZO-1, and the induction of apoptosis.

Monocrotaline pyrrole targets proteins with and without cysteine residues in the cytosol and membranes of human pulmonary artery endothelial cells.

A single injection of monocrotaline produces a pulmonary insult in rats with a phenotype similar to human primary pulmonary hypertension. Although extensively used as a model, the mechanism(s) by which this chemical insult mimics a condition with genetic and environmental links remains an enigma, although formation of protein adducts has been implicated. Monocrotaline (MCT) is non-toxic and must undergo hepatic dehydrogenation to the soft electrophile monocrotaline pyrrole as prerequisite to damaging endothelial cells lining arterioles at remote pulmonary sites. In this report we extend our earlier investigation (J. Biol. Chem. 2000, 275, 29091-29099) by examining protein adducts to lower abundance adducts, a pI range not covered before, and subcellular localization of adduct-forming proteins associated with plasma membranes. Human pulmonary artery endothelial cells were exposed to [(14)C]MCT pyrrole (MCTP) and protein targets were identified using 2-DE with IPG 4-11. Adducted proteins were identified by pI, apparent molecular weight, and PMF using MALDI-TOF MS. Results of this study show that the majority of adducts form on proteins that contain reactive thiols in a CXXC motif, such as protein disulfide isomerase A(3) (ERp57), protein disulfide isomerase (PDI), and endothelial PDI. These same proteins were the major adduct-forming proteins associated with the plasma membrane. Other proteins found to be targets were thioredoxin, galectin-1, reticulocalbin 1 and 3, cytoskeletal tropomyosin, mitochondrial ATP synthase beta-chain, annexin A2 and cofilin-1. For the first time, MCTP adducts were observed on proteins not known to contain cysteine residues. However, known reactive proteins including nucleophosmin did not form detectable adducts, potentially indicating that MCTP did not reach the interior of nucleus to the same extent as other cellular sites. These findings suggest that molecular events underlying MCTP toxicity are initiated at the plasma membrane or readily accessible subcellular regions including the cytosol and membranes of the endoplasmic reticulum and mitochondria.

Phytochemical research using accelerator mass spectrometry.

Vegetables and fruits provide an array of microchemicals in the form of vitamins and secondary metabolites (phytochemicals) that may lower the risk of chronic disease. Tracing these phytochemicals at physiologic concentrations has been hindered by a lack of quantitative sensitivity for chemically equivalent tracers that could be used safely in healthy people. Accelerator mass spectrometry is a relatively new technique that provides the necessary sensitivity (in attomoles) and measurement precision (<3%) towards 14C-labeled phytochemicals for detailed kinetic studies in humans at dietary levels.

Protein targets of 1,4-benzoquinone and 1,4-naphthoquinone in human bronchial epithelial cells.

Many aspects of the toxicity of xenobiotic compounds have been attributed to the consequences of covalent modification of specific proteins, but the nature and specificity of protein targets for classes of electrophilic toxins remain largely uncharacterized. For inhaled toxicants, the point of exposure or absorption lies with epithelial cells lining the pulmonary tree. In this study, abundant proteins in human bronchial epithelial cells that are arylated in vitro by two quinonoid compounds, 1,4-benzoquinone (BQ) and 1,4-naphthoquinone (NQ) have been detected using (14)C-labeled quinones and two-dimensional gel electrophoresis. These proteins were identified using matrix assisted laser desorption/ionization mass spectrometry for tryptic mass mapping followed by sequence database searching. Corroborative identification of protein targets was obtained from the apparent isoelectric points, molecular weights, and the use of antibody probes. There were subtle differences in the protein targets of BQ and NQ, but both associated with the following abundant proteins, nucleophosmin, galectin-1, probable protein disulfide isomerase, protein disulfide isomerase, 60 kDa heat shock protein, mitochondrial stress-70 protein, epithelial cell marker protein, and S100-type calcium binding protein A14. We further delineate the properties of these proteins that make them preferred targets and the evidence these adducts present for delivery of these quinones to subcellular compartments.

Effects of monocrotaline pyrrole and thrombin on pulmonary endothelial cell junction and matrix adhesion proteins.

Previous work in our laboratory has shown that monocrotaline pyrrole (MCTP) interacts with actin and potentiates thrombin-mediated endothelial barrier permeability through increasing the overall surface area of intercellular gaps. To better characterize endothelial barrier leak in this model, we examined the effects of MCTP and thrombin on the localization and structure of three adhesion associated proteins that directly or indirectly interact with actin in regulating barrier function: cell-cell occludens junction molecule (ZO-1), the cell-cell adherens junction linker, ss-catenin, and the cell-matrix intermediary signaling protein, focal adhesion kinase (FAK). Immunohistochemistry demonstrated that thrombin treatment resulted in radial reorganization of focal adhesions and broader distribution of adherens and occludins junctions at the cell border suggestive of membrane stretching in contracture. MCTP pretreatment resulted in fewer and more disorganized focal adhesions and marked thinning of occludins and adherens junctions. MCTP pretreatment also interfered with thrombin stimulated junctional reorganization. Western blot analysis showed thrombin stimulated catalysis of ZO-1 and FAK while MCTP pretreatment resulted in FAK fragmentation similar to previous reports for apoptosis. We conclude that both MCTP and thrombin alter critical endothelial cell adhesion molecules and this may be an underlying mechanism for the potentiating effect MCTP has on thrombin induced vascular permeability in vitro.