Incubation In Serum-free Medium Research Articles

Role of oxidative stress in vinorelbine-induced vascular endothelial cell injury

Vinorelbine (VNR), a vinca alkaloid anticancer drug, often causes vascular injury such as venous irritation, vascular pain, phlebitis, and necrotizing vasculitis. The purpose of this study was to identify the mechanisms that mediate the cell injury induced by VNR in porcine aorta endothelial cells (PAECs). PAECs were exposed to VNR for 10 min followed by further incubation in serum-free medium without VNR. The exposure to VNR (0.3–30 μM) decreased the cell viability concentration and time dependently. The incidence of apoptotic cells significantly increased at 12 h after transient exposure to VNR. At the same time, VNR increased the activity of caspases. Interestingly, VNR rapidly depleted intracellular glutathione (GSH) and increased intracellular reactive oxygen species (ROS) production. Moreover, VNR depolarized the mitochondrial membrane potential and decreased cellular ATP levels. These VNR-induced cell abnormalities were almost completely inhibited by GSH and N-acetylcysteine. On the other hand, l-buthionine-( S,R)-sulfoximine, a specific inhibitor of GSH synthesis, aggravated the VNR-induced loss of cell viability. These results clearly demonstrate that VNR induces oxidative stress by depleting intracellular GSH and increasing ROS production in PAECs, and oxidative stress plays an important role in the VNR-induced cell injury.

Lysophosphatidylcholine Exhibits Selective Cytotoxicity, Accompanied by ROS Formation, in RAW 264.7 Macrophages

Lysophosphatidylcholine (lysoPtdCho) is a component of oxidized low density lipoprotein, and is involved in the pathogenesis of atherosclerosis and inflammation. We studied the effects of lysoPtdCho on cytotoxicity, reactive oxygen species (ROS) production, activation of the extracellular signal-regulated kinase (ERK), mitogen-activated protein kinases and pro-inflammatory gene expression in RAW 264.7 murine macrophage cells. When cells were exposed to lysoPtdCho with various acyl chains in a culture medium containing 10% fetal bovine serum, only 1-linoleoyl (C18:2) lysoPtdCho showed a remarkable cytotoxicity, reaching the highest level at 24 h, and elicited ROS production, suggesting that oxidative stress might be implicated in the cytotoxicity of 1-linoleoyl (C18:2) lysoPtdCho. Presumably in support of this, antioxidants such as magnolol or trolox prevented 1-linoleoyl (C18:2) lysoPtdCho-induced cytotoxicity as well as ROS production, although only partially. Furthermore, the phosphorylation of ERK 1/2 and the expression of pro-inflammatory cytokines such as IL-1beta, CCL2 and CCL5 were augmented by 1-linoleoyl (C18:2) lysoPtdCho. Meanwhile, there was no structural importance of the acyl chain for the cytotoxic action of lysoPtdCho during 10 min incubation in serum-free media. Taken together, it is suggested that in a serum-containing medium, 1-linoleoyl (C18:2) lysoPtdCho can cause a significant cytotoxicity through ROS production, probably accompanied by activation of ERK and induction of related inflammatory cytokines, in RAW 264.7 cells.

Graft-Vs-Graft Immune Interaction Is the Likely Mechanism of Absolute Unit Dominance in Double Unit Cord Blood (DCB) Transplantation Using Patient DCB Grafts: An in Vivo but Not in Vitro Phenomenon

DCB transplantation (DCBT) appears to enhance engraftment despite sustained hematopoiesis usually being derived from a single unit. Therefore, to further investigate DCB biology we established in vitro and in vivo DCB models using samples from patient DCB grafts.METHODS: Patients with high-risk hematologic malignancies were transplanted with DCB grafts and consented to ≤5% of each unit to be used for research purposes. We used mononuclear cells (MNC) or MicroBead selected CD34+ cells (85–90% enrichment) to assess short-term (2 week) progenitor colony-forming units (CFU) and long-term (5 week) stem cell (cobblestone-area-forming-cells, CAFC) potential of each unit alone and in DCB combination. In CFU assays cells were cultured immediately with G-CSF, KL, IL-3 and erythropoietin, or after overnight incubation in serum-free media with Flt3L, KL, and TPO. CAFC assays were performed using MS5 stroma. In addition, sublethally irradiated NOD/SCID IL2R-γ null (NOG) mice were transplanted with MNC or CD34+ via IV injection in the same ratio as transplanted into the patients. In all DCB assays the contribution of each unit was measured by qPCR for informative short tandem repeat (STR) regions and correlated with the engrafting (winning) unit in the patient.RESULTS: All DCBT recipients studied (n=35) engrafted with a single unit except one patient who had sustained co-engraftment of both units. In CFU assays with MNC (n=5) or CD34+ (n=5) CFU were seen in winner cultured alone, loser alone, and with DCB. CFU number was not superior with winner alone and there was no augmentation of CFU with DCB. CFU DCB assays were a mix of both units with in vitro unit proportions concordant with the number of colonies formed by each unit alone (mean % winner in DCB ±SEM: 53%±12) with no correlation with the clinical winner. Further, overnight incubation did not significantly alter CFU numbers or unit proportions in DCB co-culture (54%±10). In CAFC assays with MNC (n=4) or CD34+ (n=5) CAFC were also present in all 3 conditions, not significantly superior in the winner, and there was no CAFC augmentation with DCB. CAFC in DCB assays were mixed in 6/9 and a single unit in 3/9 using either CD34+ or MNC with no correlation with the winner in the patient. Further, unlike the CFU assays, there was no concordance between CAFC number when each unit cultured alone and the in vitro unit dominance with DCB co-culture. The clinical winner was not predicted by the calculated number of infused CFU or CAFC to the patient. Murine experiments (n=27) included 19 with and 8 without DCB; using MNC in 20 and CD34+ in 7 (including 2/7 with CD34+ DCB plus CD34− add-backs). All mice engrafted with either unit alone or DCB at 4–7 weeks. Engraftment was not superior with the clinical winner alone (n=27) or in DCB (n=19).Table: NOG Mice Engraftment by STR in DCBT of MNC or CD34+Infused Cell TypeNSingle Unit Dominance 100%Correlation with Winner in PatientMNC DCB1211/12 *Mimicked patient in 11/12 (92%)*CD34+ DCB74/7Mimicked patient in 2/7 (29%)CD34+ DCB + CD34− unit#1CD34+ DCB + CD34− unit#222/2Winner dictated by origin of CD34-fraction* The single MNC experiment with mixed chimerism mimicked patient engraftment as this was the single patient with sustained co-engraftment of both units. However, in another MNC experiment (1/12) mice had single unit dominance but with the losing unit in the patient.CONCLUSION: in vitro CFU and CAFC DCB assays were predominantly mixed with no correlation with the clinical winner. However, as seen in human DCBT, murine MNC DCBT mimicked patient engraftment (11/12) with absolute single unit dominance (11/12). Notably, single unit dominance and clinical correlation was lost with CD34+ selection. Further, absolute unit dominance was restored by the add-back of the CD34− fraction. This is strong support that absolute unit dominance in vivo is linked to a graft-vs-graft immune interaction. Further experiments using this murine model are underway which promise to further elucidate DCB biology.

(22R,23R)-3β-hydroxy-22,23-epoxy-5α-ergost-8(14)-en-15-one: Improved synthesis and toxicity to MCF-7 cells

The chemical synthesis of (22R,23R)-3beta-hydroxy-22,23-epoxy-5alpha-ergost-8(14)-en-15-one from (22E)-3beta-acetoxy-5alpha-ergosta-7,14,22-triene was improved. The stages of obtaining and isomerizing (22E)-3beta-acetoxy-14alpha,15alpha-epoxy-5alpha-ergosta-7,22-diene were optimized. The introduction of the (22R,23R)-epoxide cycle was carried out by a basic treatment of intermediate (22S,23R)-3beta,23-diacetoxy-22-iodo-5alpha-ergost-8(14)-en-15-one. In cells of human mammary gland carcinoma MCF-7 (22R,23R)-3beta-hydroxy-22,23-epoxy-5alpha-ergost-8(14)-en-15-one showed a high toxicity (TC(50) = 0.4 +/- 0.1 microM for 48-h incubation in serum-free medium).

Interleukin-4 induces the activation and collagen production of cultured human intrahepatic fibroblasts via the STAT-6 pathway

Interleukin-4 (IL-4) is overexpressed in liver grafts in a context of severe recurrent hepatitis C, during which the development of fibrosis is dramatically accelerated. In this study, we examined the effects of IL-4 on the activation and collagen production of cultured human intrahepatic (myo)fibroblasts (hIHFs), and investigated the underlying mechanisms. The myofibroblastic nature of cells was evaluated morphologically using activation markers (smooth muscle α-actin, vimentin and prolyl 4-hydroxylase). Quiescent hIHFs were obtained by cell incubation in serum-free medium or cell culture on Matrigel. We first analyzed IL-4 receptor expression, STAT-6 activation by IL-4, and STAT-6 inhibition by an anti-IL-4 antibody or by STAT-6 small-interfering RNA (siRNA) transfection. We then focused on collagen production, using quantitative real-time PCR to analyze the effect of IL-4 on the mRNA expression of collagens I, III and IV, and on collagen levels in supernatants of hIHFs, using the Sircol collagen assay. hIHFs cultured in plastic wells appeared to be morphologically activated. The expression of activation markers was reduced by serum deprivation or culture on Matrigel, and restored by IL-4 incubation. The IL-4 receptor was expressed by hIHFs, and STAT-6 was activated following incubation with IL-4. Both anti-IL-4 antibody and STAT-6 siRNA transfection inhibited this activation. The treatment of hIHFs with IL-4 increased the mRNA expression of collagens I, III and IV (P<0.05) and elevated collagen levels in supernatants (P=0.01 vs untreated cells). Therefore, IL-4 exerts profibrotic effects by activating hIHFs and inducing collagen production and secretion. This effect requires IL4-R binding and STAT-6 activation. IL-4 may thus be involved in accelerated course of fibrogenesis during recurrent hepatitis C.

Hepatocyte-specific c-Met Deletion Disrupts Redox Homeostasis and Sensitizes to Fas-mediated Apoptosis

The hepatocyte growth factor and its receptor c-Met direct a pleiotropic signal transduction pathway that controls cell survival. We previously demonstrated that mice lacking c-Met (Met-KO) in hepatocytes were hypersensitive to Fas-induced liver injury. In this study, we used primary hepatocytes isolated from Met-KO and control (Cre-Ctrl) mice to address more directly the protective effects of c-Met signaling. Loss of c-Met function increased sensitivity to Fas-mediated apoptosis. Hepatocyte growth factor suppressed apoptosis in Cre-Ctrl but not Met-KO hepatocytes concurrently with up-regulation of NF-kappaB and major antiapoptotic proteins Bcl-2 and Bcl-xL. Intriguingly, Met-KO hepatocytes exhibited intrinsic activation of NF-kappaBas well as Bcl-2 and Bcl-xL. Furthermore, unchallenged Met-KO cells displayed oxidative stress as evidenced by overproduction of reactive oxygen species, which was associated with greater NADPH and Rac1 activities, was blocked by the known NADPH oxidase inhibitors, and was paralleled by increased lipid peroxidation and reduced glutathione (GSH) content. N-Acetylcysteine, an antioxidant and GSH precursor, significantly reduced Jo2-induced cell death. Conversely, the GSH-depleting agent buthionine sulfoximine completely abolished the protective effects of N-acetylcysteine in Met-KO hepatocytes. In conclusion, genetic inactivation of c-Met in mouse hepatocytes caused defects in redox regulation, which may account for the increased sensitivity to Fas-induced apoptosis and adaptive up-regulation of NF-kappaB survival signaling. These data provide evidence that intact c-Met signaling is a critical factor in the protection against excessive generation of endogenous reactive oxygen species.

Instability of self‐assembled monolayers as a model material system for macrophage/FBGC cellular behavior

Novel self-assembled monolayers (SAMs) designed to present homogenous surface chemistries were utilized to further investigate the material surface chemistry dependent macrophage and foreign-body giant cell (FBGC) behaviors, including macrophage adhesion, fusion, and apoptosis. Contact angle analysis revealed instabilities in the --CH(3) and --COOH terminated SAM surfaces upon incubation in serum-free media (SFM) at 37 degrees C or under dry, room temperature conditions. Further analysis indicated that the --CH(3) terminated SAM surface degraded rapidly within 2 h and loss of sufficient SAM units to be comparable to the gold (Au) control surface, within 24 h of incubation in SFM at 37 degrees C. After 5 days of incubation in SFM at 37 degrees C, the contact angles for the --COOH terminated SAM surfaces increased markedly. AFM analysis confirmed the desorption of --CH(3) terminated SAM molecules from the surface with increased roughness and marked appearance of peaks and valleys within 2 h. A decrease in the thickness of the --COOH terminated SAM surface also suggests molecular desorption over time. No significant changes in contact angle or AFM analyses were observed on the --OH terminated SAM surfaces. Cellular adhesion decreased more rapidly on the Au control and --CH(3) terminated SAM surfaces in comparison to the other surfaces. However by day 10, cellular adhesion, fusion, and apoptosis were comparable on all SAM surfaces and the Au control. These studies suggest that SAM surfaces may not be suitable for long-term studies where material dependent properties are investigated.

Effects of tibolone and its metabolites on Angiopoietin-1, Tie-2 and tumor necrosis factor-α mRNA in Ishikawa cells: Implication for tibolone's effects on the endometrium

Objective To determine the effect of 17β-estradiol, medroxyprogesterone acetate, tibolone and tibolone metabolites on Angiopoietin-1, Tie-2, and tumor necrosis factor-α in Ishikawa cells, in vitro. We hypothesized that differential effects on angiogenic factors or inflammatory cytokines by individual hormones may be related to the endometrial bleeding in postmenopausal women using hormone therapy. Design Ishikawa cells were cultured to 80% confluence, in vitro. After 24 h incubation in serum-free media, 1.0, 0.1 and 0.01 μM of 17β-estradiol, medroxyprogesterone acetate, tibolone, 3α-hydroxytibolone, 3β-hydroxytibolone, and Δ 4-tibolone were added to the Ishikawa cells. The cells plus steroids were then incubated for a further 24 h. Total RNA was extracted from control and treated Ishikawa cells. After reverse transcription, Angiopoietin-1, Tie-2, tumor necrosis factor-α, and β-actin cDNAs were amplified in a polymerase chain reaction spiked with 33p-dCTP. Relative abundance of Angiopoietin-1, Tie-2, and tumor necrosis factor-α mRNA was measured by scintillation spectroscopy. Results 17β-Estradiol and medroxyprogesterone acetate increased Angiopoietin-1 mRNA significantly higher than control, tibolone and tibolone hydroxy metabolites. Δ 4-Tibolone at all concentrations tested did not increase Angiopoietin-1. None of the steroids tested at any concentration altered Tie-2 mRNA expression compared to control. 17β-Estradiol at 1.0 and 0.1 μM increased tumor necrosis factor-α mRNA significantly higher than control. Medroxyprogesterone acetate only at 1.0 μM increased tumor necrosis factor-α mRNA above control levels. Tibolone, 3α-hydroxytibolone, 3β-hydroxytibolone, and Δ 4-tibolone at every concentration had no effect on tumor necrosis factor-α mRNA abundance. Conclusions Δ 4-Tibolone did not stimulate Angiopoietin-1, while the other steroids had differential effects greater than control. None of the steroids changed the expression of Tie-2 mRNA. Tumor necrosis factor-α was increased by 17β-estradiol and by the highest concentration of medroxyprogesterone acetate. We interpret these results as supportive of our hypothesis that differential effects on angiogenic factors or inflammatory cytokines by individual steroids may be related to the clinical occurrence of endometrial bleeding in postmenopausal women using hormone therapy.

Regulation of NF-κB-dependent Gene Expression by the POU Domain Transcription Factor Oct-1

Maintenance of the cells of the vessel wall in a quiescent state is an important aspect of normal vascular physiology. Transcriptional repressors are widely believed to regulate this process, yet the exact factors involved and the mechanism of repression are not known. Here, we report that the POU domain transcription factor Oct-1 represses the expression of E-selectin and vascular cell adhesion molecule (VCAM-1), two cytokine-inducible, NF-kappaB-dependent endothelial-leukocyte adhesion molecules that participate in the leukocyte recruitment phase of the inflammatory response. Co-transfection and microinjection studies demonstrate that Oct-1 blocks tumor necrosis factor alpha-stimulated E-selectin and VCAM-1 expression. Gene expression arrays indicate that control of tumor necrosis factor alpha-induced, NF-kappaB-dependent gene expression by Oct-1 is promoter-specific. A DNA-binding mutant of Oct-1 represses NF-kappaB-dependent reporter gene expression. Biochemically, Oct-1 interacts with p65, suggesting that Oct-1 is involved in the regulation of NF-kappaB transactivation function. NF-kappaB-dependent gene expression is more pronounced in Oct-1-deficient than in wild-type murine embryonic fibroblasts, and reintroduction of human Oct-1 abolishes these differences. Finally, the cytokine interleukin-6 induces Oct-1 gene expression, providing a biologically relevant means by which NF-kappaB-dependent gene expression can be selectively reverted by Oct-1 to quiescent levels.

Effects of 17?-estradiol, progesterone, synthetic progestins, tibolone, and raloxifene on vascular endothelial growth factor and Thrombospondin-1 messengerRNA in breast cancer cells

To determine the effect of 17beta-estradiol, raloxifene, progesterone, medroxyprogesterone acetate (MPA), levonorgestrel (LNG), norethindrone (NET), tibolone and tibolone metabolites on vascular endothelial growth factor (VEGF) isoforms 121 and 165 and Thrombospondin-1 (TSp-1) messenger RNA (mRNA) in two breast cancer cell lines, MCF-7 and T47-D. MCF-7 and T47-D cells were cultured to 80% confluence, in vitro. After 24 h incubation in serum-free media, 1.0, 0.1, and 0.01 muM of 17beta-estradiol, raloxifene, raloxifene plus ICI 182780, tibolone, 3alpha-hydroxytibolone, and 3beta-hydroxytibolone were added to MCF-7 cells. Progesterone, MPA, LNG, NET, and Delta(4) tibolone at 1.0, 0.1, and 0.01 muM were added to T47-D cells. The cells plus steroids were incubated for a further 24 h. Total RNA was isolated using TRIZOL and reverse transcriptase-polymerase chain reaction was carried out using primers for VEGF, TSP-1, and cyclophilin, the latter as an internal control. Semiquantitative analysis was performed using 33P-CTP for radioactive labeling during the polymerase chain reaction. 17beta-estradiol, raloxifene, tibolone, 3alpha-hydroxytibolone, and 3beta-hydroxytibolone had no effect on VEGF mRNA in MCF-7 cells. Progesterone, MPA, LNG, and NET increased VEGF mRNA in T47-D cells. Delta(4) tibolone also increased VEGF mRNA but to a lesser extent than the progestogens. Raloxifene increased TSP-1 mRNA, this effect was not reversed by the addition of ICI 182780 to the media. 17beta-estradiol, raloxifene, tibolone and tibolone hydroxy-metabolites had no effect on VEGF mRNA in MCF-7 cells. Progesterone and progestins increased VEGF mRNA in T47-D breast cancer cells. Delta(4) tibolone was less effective than progestogens on this angiogenic gene in the T47-D cells. Raloxifene increased TSP-1. These differential effects may be related to breast cancer growth.

Insulin Reverses Growth Hormone-induced Homologous Desensitization

Growth hormone (GH) is secreted in a pulsatile pattern to promote body growth and metabolism. GH exerts its function by activating several signaling pathways, including JAK2/STAT and MEK/ERK. ERK1/2 activation by GH plays important roles in gene expression, cell proliferation, and growth. We previously reported that in rat H4IIE hepatoma cells after an initial GH exposure, a second GH exposure induces STAT5 phosphorylation but not ERK1/2 phosphorylation (Ji, S., Frank, S. J., and Messina, J. L. (2002) J. Biol. Chem. 277, 28384-28393). In this study the mechanisms underlying GH-induced homologous desensitization were investigated. A second GH exposure activated the signaling intermediates upstream of MEK/ERK, including JAK2, Ras, and Raf-1. This correlated with recovery of GH receptor levels, but was insufficient for GH-induced phosphorylation of MEK1/2 and ERK1/2. Insulin restored the ability of a second GH exposure to induce phosphorylation of MEK1/2 and ERK1/2 without altering GH receptor levels or GH-induced phosphorylation/activation of JAK2 and Raf-1. GH and insulin synergized in promoting cell proliferation. Further investigation suggested that insulin increased the amount of MEK bound to KSR (kinase suppressor of Ras) and restored GH-induced tyrosine phosphorylation of KSR. Previous GH exposure also induced desensitization of STAT1 and STAT3 phosphorylation, but this desensitization was not reversed by insulin. Thus, insulin-regulated resensitization of GH signaling may be necessary to reset the complete response to GH after a normal, physiologic pulse of GH.

The Early-Immediate Gene EGR-1 Is Induced by Transforming Growth Factor-β and Mediates Stimulation of Collagen Gene Expression

Transforming growth factor-beta (TGF-beta) stimulates collagen synthesis and accumulation, and aberrant TGF-beta signaling is implicated in pathological organ fibrosis. Regulation of type I procollagen gene (COL1A2) transcription by TGF-beta involves the canonical Smad signaling pathway as well as additional protein and lipid kinases, coactivators, and DNA-binding transcription factors that constitute alternate non-Smad pathways. By using Affymetrix microarrays to detect cellular genes whose expression is regulated by Smad3, we identified early growth response factor-1 (EGR-1) as a novel Smad3-inducible gene. Previous studies implicated Egr-1 in cell growth, differentiation, and survival. We found that TGF-beta induced rapid and transient accumulation of Egr-1 protein and mRNA in human skin fibroblasts. In transient transfection assays, TGF-beta stimulated the activity of the Egr-1 gene promoter, as well as that of a minimal Egr-1-responsive reporter construct. Furthermore, TGF-beta enhanced endogenous Egr-1 interaction with a consensus Egr-1-binding site element and with GC-rich DNA sequences of the human COL1A2 promoter in vitro and in vivo. Forced expression of Egr-1 by itself caused dose-dependent up-regulation of COL1A2 promoter activity and further enhanced the stimulation induced by TGF-beta. In contrast, the TGF-beta response was abrogated when the Egr-1-binding sites of the COL1A2 promoter were mutated or deleted. Furthermore, Egr-1-deficient embryonic mouse fibroblasts showed attenuated TGF-beta responses despite intact Smad activation, and forced expression of ectopic EGR-1 in these cells could restore COL1A2 stimulation in a dose-dependent manner. Taken together, these findings identify Egr-1 as a novel intracellular TGF-beta target that is necessary for maximal stimulation of collagen gene expression in fibroblasts. The results therefore implicate Egr-1 in the profibrotic responses elicited by TGF-beta and suggest that Egr-1 may play a new and important role in the pathogenesis of fibrosis.

Promoter Organization of the Interferon-A Genes Differentially Affects Virus-induced Expression and Responsiveness to TBK1 and IKK∈

Virus-induced expression of interferon (IFN)-A genes is regulated by two members of the IFN regulatory factor (IRF) family, IRF-3 and IRF-7, which are activated by phosphorylation during viral infection by the IKK-related serine/threonine kinases TBK1 and IkappaB kinase epsilon (IKKepsilon). In this study, we demonstrate that three IRF-binding sites located in the virus-responsive element mediate the transcriptional activation of the IFN-A4 promoter by IRF-3. The precise arrangement of these IRF elements is required for synergistic activation of the IFN-A4 promoter following Newcastle disease virus infection or activation by TBK1 or IKKepsilon. The ordered assembly of IRF-3 multimers on the promoter also determines cooperative recruitment of IRF-3 and CREB-binding protein and differential virus-induced expression of IFN-A4 gene promoter compared with IFN-A11. Naturally occurring nucleotide substitutions disrupt two of the IRF elements in the IFN-A11 gene promoter, leading to a dramatic decrease in IRF-3 and CREB-binding protein recruitment and in IRF-3-dependent transcription. Transcription of the IFN-A4 promoter by IRF-7 is mediated by two IRF elements; promoter mutants that carry a reversed IRF element retain the ability to respond to IKKepsilon or TBK1 expression in the presence of IRF-7 but lose the capacity to respond to virus or kinase-induced IRF-3. Interestingly, IKKepsilon or TBK1 stimulates the IRF-7-mediated transcription of IFN-A11, although at a lesser extent compared with IFN-A4. Our data indicate that virus-induced expression of IFN-A genes is dictated by the organization of IRF elements within the IFN-A promoters and that the differential IFN-A gene expression, based on the IRF-3 responsiveness, is partially compensated in the presence of IRF-7 when both factors are activated by IKKepsilon or TBK1.

Natural nutrient mixture effectively reduces collagen matrix contraction driven by human uterine smooth muscle cells

Abnormal uterine myometrial contractility causes preterm delivery, contributing to perinatal morbidity and mortality. Disturbances in hormonal regulation and inflammation-related processes have been attributed a role in the pathophysiological mechanisms of uterine contractility. We investigated the effects of natural nutrients on uterine tissue contractility in vitro. We used an in vitro model of collagen I gel contraction driven by embedded cultured human uterine smooth muscle cells (SMC). The effects of tested compounds were evaluated using their capacity to affect gel contraction (measured by reduction in gel area during 24-h incubation in serum free medium). Cellular expression of matrix metalloproteinases (MMP) was followed by gel zymography. Collagen gel contraction driven by uterine SMC was significantly stimulated by potassium chloride, pituitary hormone oxytocin and by inflammatory cytokine alpha-tumor necrosis factor (TNF-alpha). Accelerated gel contraction was accompanied by elevated secretion of MMP-2 and MMP-9 into cell culture media. Among a variety of purified bioflavonoids and polyphenol-enriched plant extracts tested for their ability to counteract uterine SMC-dependent collagen gel contraction, the strongest effects were demonstrated by epigallocatechin gallate and green tea leaf extract, respectively. The addition of ascorbic acid and the amino acids lysine, arginine, cysteine and proline to green tea extract further increased its effectiveness. A reduction in gel contraction correlated with decreased MMP expression. Based on these findings, we found that nutrients can effectively counteract uterine myometrial contraction and MMP activity in vitro, suggesting that pathophysiological mechanisms of abnormal uterine myometrial contractility can be counteracted by a combination of naturally occurring nutrients. These mechanisms might involve extracellular matrix remodeling.

Deferasirox (Exjade®, ICL670): A Journey into Labile Iron Centers of Living Cardiomyocytes.

Introduction and Aims: Iron toxicity that prevails in iron overload is associated with forms of labile cell iron (LCI) that appear in tissues such as heart, endocrine glands and liver. The primary goal of chelation is to reduce LCI appearance by preventing the entry of labile plasma iron into cells and by chelating LCI. The present study was aimed at evaluating the capacity of the novel oral iron chelator deferasirox (a) to access cardiomyocytes and chelate LCI in the cell organelles harboring labile iron and thereby prevent its involvement in reactive oxidant species (ROS) formation and (b) to reduce the cell iron load by extraction of labile and accumulated iron.Methods: LCI pools were revealed by fluorescence microscopy imaging using novel fluorescent iron sensors addressed to various organelles (cytosol, nucleus, mitochondria and endosomeslysosomes) via specific peptide target sequences. Resident or imported labile iron binds the fluorescence metal sensor and quench its fluorescence. The chelator's capacity to restore quenched fluorescence in a given cell compartment is indicative of its permeation and chelating potential, which is also assessed with ROS-sensitive probes targeted to cytosol or mitochondria (Glickstein et al 2005, Blood In press). Chelator-mediated extraction of cell iron was assessed in cardiomyocytes from neonatal rats or murine H9C2 cells preincubated with 360μM 59Fe-ferric ammonium citrate for 3 h or 24 h (representing respectively radiolabelled LCI=RLCI or stored iron=RSI). Iron extraction was followed as cell iron retention following 0.5–24 h treatment with chelators in culture medium (with 20% fetal calf serum ≡ 1% albumin or with 4% bovine serum albumin).Results and Discussion: In situ fluorescence LCI tracing studies indicate that LCI chelation by up to 100 μM deferoxamine (DFO) (x10 higher than normally attained in plasma during infusion) demanded incubation times >1 h. Conversely, the relatively smaller and more lipophilic deferasirox at 50–100 μM demonstrably gained access to all LCI pools associated with organelles following 20′–30′ incubation in serum-free medium and 30′–60′ in serum-containing medium (note: deferasirox reaches clinically Cmax and trough plasma levels of approximately 100 μM and 20 μM respectively with daily doses of 20 mg/kg). Following 6 h treatment with chelator in serum-containing medium, LCI (measured as RLCI) was 15 and 60% reduced by 20 and 100 μM DFO respectively, and 15 and 34% reduced by 20 and 100 μM deferasirox, respectively. On the other hand, the reduction in mostly stored RSI with 20 and 100 μM chelator was 5 and 45% for DFO and 0 and 20% by deferasirox, respectively.Conclusions: At drug concentrations equivalent to those attained in plasma with a single daily drug intake, ie 50–100 μM: (a) deferasirox gains relatively fast entry into cardiomyocytes and their intracellular compartments, scavenges LCI and attenuates ROS formation; (b) exit of the (deferasirox)2-Fe chelates formed from LCI pools (RLCI) by added drug is relatively slower than entry of the free drug, but evident within 1–3 h even at 20 μM drug concentrations (equivalent to trough plasma levels).

Inhibition of Either Phosphatidylinositol 3-kinase/Akt or the Mitogen/Extracellular-regulated Kinase, MEK/ERK, Signaling Pathways Suppress Growth of Breast Cancer Cell Lines, but MEK/ERK Signaling is Critical for Cell Survival

The phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathways are important integrators of growth and survival signals originating from extracellular stimuli. We assessed the importance of these signaling pathways in the growth and survival of 8 breast cell lines (MCF10A, an immortalized line; and 7 cancer cell lines). The cell lines expressed variable levels of both phosphorylated ERK and phosphorylated Akt, but these were unchanged by incubation in serum-free medium. Despite continued activity of these pathways, the cells arrested growth in the absence of serum demonstrating that additional pathways are required for growth. Incubation with the PI3K inhibitor LY294002 suppressed growth of all cell lines, but most remained viable for at least 7-14 days. This long-term survival may be attributable to recovery of phospho-Akt by 24-48 h despite the continued presence of active LY294002, suggesting that alternate pathways may be activating Akt. In contrast, incubation with the MEK inhibitor U0126 not only arrested growth, but also killed all the cell lines within 2-4 days in the absence of serum; the presence of serum only slighted extended viability, except in MCF10A and MDA-MB-468 cells, in which serum provided significantly greater protection. It is likely that these signaling pathways control the level of pro-and anti-apoptotic proteins, yet assessment of Bcl-2 and Bcl-X showed dramatic reduction in level only when large numbers of cells were dead suggesting this may be a consequence rather than cause of death. Overall, the results demonstrate that the MEK/ERK pathway represents the more critical pathway for cell survival of these breast cancer cell lines, and suggest this pathways represents the better target for cancer therapy.

Effect of 17β-estradiol, progesterone, synthetic progestins, tibolone, and tibolone metabolites on vascular endothelial growth factor mRNA in breast cancer cells

To determine the effect of 17beta-estradiol, progesterone, medroxyprogesterone acetate, levonorgestrel, norethindrone, tibolone, and tibolone metabolites on vascular endothelial growth factor (VEGF) isoforms 121 and 165 mRNA in two breast cancer cell lines, MCF-7 (estrogen receptor rich) and T47-D (progesterone receptor rich), in vitro. Prospective basic research study. Basic research laboratory. None. MCF-7 and T47-D cells were cultured to 80% confluence in vitro. After 24 hours' incubation in serum-free media, 1.0, 0.1, and 0.01 microM of 17beta-estradiol, tibolone, 3alpha-hydroxytibolone and 3beta-hydroxytibolone were added to MCF-7 cells. Progesterone, medroxyprogesterone acetate, levonorgestrel, norethindrone, and Delta4 tibolone at 1.0, 0.1, and 0.01 microM were added to T47-D cells. The cells plus steroids were incubated for a further 24 hours. Isolation and identification of VEGF isoforms 121 and 165 using semiquantitative polymerase chain reaction, gel electrophoresis, with cyclophilin as an internal control. 17beta-estradiol, tibolone, 3alpha-hydroxytibolone, and 3beta-hydroxytibolone had no effect on VEGF mRNA in MCF-7 cells. Progesterone, medroxyprogesterone acetate, levonorgestrel, and norethindrone increased VEGF mRNA in T47-D cells. Delta4-Tibolone also increased VEGF mRNA but to a lesser extent than the progestogens. 17beta-estradiol, tibolone, and tibolone hydroxy-metabolites had no effect on VEGF mRNA in MCF-7 cells. Progesterone and progestins increased VEGF mRNA in T47-D breast cancer cells, but Delta4-tibolone was less effective than progestogens on this angiogenic gene in the T-47 D cells. This differential effect may be related to breast cancer growth.

C5L2 Is a Functional Receptor for Acylation-stimulating Protein

C5L2 binds acylation-stimulating protein (ASP) with high affinity and is expressed in ASP-responsive cells. Functionality of C5L2 has not yet been demonstrated. Here we show that C5L2 is expressed in human subcutaneous and omental adipose tissue in both preadipocytes and adipocytes. In mice, C5L2 is expressed in all adipose tissues, at levels comparable with other tissues. Stable transfection of human C5L2 cDNA into HEK293 cells results in ASP stimulation of triglyceride synthesis (TGS) (193 +/- 33%, 5 microM ASP, p < 0.001, where basal = 100%) and glucose transport (168 +/- 21%, 10 microM ASP, p < 0.001). C3a similarly stimulates TGS (163 +/- 12%, p < 0.001), but C5a and C5a des-Arg have no effect. The ASP mechanism is to increase Vmax of glucose transport (149%) and triglyceride (TG) synthesis activity (165%) through increased diacylglycerolacyltransferase activity (200%). Antisense oligonucleotide down-regulation of C5L2 in human skin fibroblasts decreases cell surface C5L2 (down to 54 +/- 4% of control, p < 0.001, comparable with nonimmune background). ASP response is coordinately lost (basal TGS = 14.6 +/- 1.6, with ASP = 21.0 +/- 1.4 (144%), with ASP + oligonucleotides = 11.0 +/- 0.8 pmol of TG/mg of cell protein, p < 0.001). In mouse 3T3-L1 preadipocytes, antisense oligonucleotides decrease C5L2 expression to 69.5 +/- 0.5% of control, p < 0.001 (comparable with nonimmune) with a loss of ASP stimulation (basal TGS = 22.4 +/- 2.9, with ASP = 39.6 +/- 8.8 (177%), with ASP + oligonucleotides = 25.3 +/- 3.0 pmol of TG/mg of cell protein, p < 0.001). C5L2 down-regulation and decreased ASP response correlate (r = 0.761, p < 0.0001 for HSF and r = 0.451, p < 0.05 for 3T3-L1). In HEK-hC5L2 expressing fluorescently tagged beta-arrestin, ASP induced beta-arrestin translocation to the plasma membrane and formation of endocytic complexes concurrently with increased phosphorylation of C5L2. This is the first demonstration that C5L2 is a functional receptor, mediating ASP triglyceride stimulation.

Albumin and urea production by hepatocytes cultured on extracellular matrix proteins-conjugated poly (vinyl alcohol) membranes

Production of albumin and urea by mouse hepatocytes on poly(vinylalcohol-co-ethylamine) (PVA-EA) membranes containing immobilized extracellular matrix (ECM) proteins was investigated for 7 days. The amount of ECM proteins (collagen, vitronectin and laminin) immobilized on PVA-EA and PVA-ECM membranes was determined to range from 1.09 μg/cm2 to 1.60 μg/cm2. Hepatocytes cultured on PVA-ECM membranes in serum-free media showed higher albumin production than those cultured on PVA-EA membranes after a 7-day incubation under the conditions in this study. Urea production by hepatocytes on PVA-ECM membranes was also determined to be higher than that on PVA-EA membranes up until day 5 of incubation in serum-free media, whereas no difference of urea production by hepatocytes on different PVA-ECM membranes and PVA-EA membranes was observed at 7 days of incubation. The effect of ECM proteins in PVA-ECM membranes on hepatocyte function (such as albumin and urea production) was observed in hepatocytes cultured in serum-free media up to day 5 of incubation. The ECM proteins immobilized on the PVA-ECM membranes contributed not only to the long-term stable production of albumin and urea by hepatocytes, but also the improved survival (viability) of hepatocytes on PVA-ECM membranes.

Characteristics of cellular proliferation in the developing human retina.

The expression of proliferation-associated proteins Ki67 and PCNA was studied in the retinal rudiments of human embryos at 5-8 weeks of development; studies also addressed the numbers of nucleoli in the nuclei of neuroepithelial cells (with consideration of their distances to the apical surface) and DNA-synthesizing cells after transient (20 min) in vitro incubation in serum-free medium containing BrdU. The retinal rudiment of embryos at five weeks of development had neuroepithelium of the typical structure. BrdU-positive nuclei and nuclei with small numbers of nucleoli were located in the basal part of the ventricular zone. However, this organization was disrupted during the initial period of formation of the inner nuclear layer (six weeks). At this time, DNA-synthesizing cells were found even at the apical surface. Retinal rudiments of embryos at 6-7 weeks of development contained an additional area of cell proliferation in the Chievitz layer and the inner nuclear layer. In eight-week embryos, dividing cells were located in the outer nuclear layer, which again acquired the organization typical of neuroepithelium.