sepharose and Hypoxia

Hypoxia in local tumors leads to the failure or resistance of radiotherapy (RT) and high-dose RT will cause systemic reactions and local radiation damage. As a non-chemotherapeutic intervention, photothermal therapy (PTT) can remove tumor tissues through thermal ablation as well as effectively improve the microenvironment of hypoxic cells. Therefore, the combined use of PTT and RT (thermoradiotherapy) has urgently become an efficient treatment. In this work, by encapsulating prussian blue (PB) nanoparticles in agarose hydrogel, we developed an injectable hybrid light-controlled hydrogel system as a PB reservoir and release controller (PRC) which can realize single injection and multiple treatments in vivo. Under the irradiation of 808 nm near-infrared (NIR) laser, PB nanoparticles convert laser energy into heat energy, causing degradation of agarose hydrogel and the release of PB nanoparticles. Due to the excellent photothermal properties of PB, photothermal treatment in the NIR Biological Windows can greatly enhance the sensitivity of tumor cells to RT. Meanwhile, PB nanoparticles can also be a nanozyme to drive the decomposition of endogenous hydrogen peroxide (H

Topics: Cell Line, Tumor; Humans; Hydrogels; Hydrogen Peroxide; Hypoxia; Nanoparticles; Oxygen; Phototherapy; Sepharose

Degeneration of the intervertebral discs is strongly implicated as a cause of low back pain. Since current treatments for discogenic low back pain show poor long-term efficacy, a number of new biological strategies are being pursued. For such therapies to succeed, it is critical that they be validated in conditions that mimic the unique biochemical microenvironment of the nucleus pulposus (NP), which include low oxygen tension. Therefore, the objective of this study was to investigate the effects of oxygen tension on NP cell functional extracellular matrix elaboration in 3D culture. Bovine NP cells were encapsulated in agarose constructs and cultured for 14 or 42 days in either 20% or 2% oxygen in defined media containing transforming growth factor beta-3. At each time point, extracellular matrix composition, biomechanics, and mRNA expression of key phenotypic markers were evaluated. Results showed that while bulk mechanics and composition were largely independent of oxygen level, low oxygen promoted improved restoration of the NP phenotype, higher mRNA expression of extracellular matrix and NP specific markers, and more uniform matrix elaboration. These findings indicate that culture under physiological oxygen levels is an important consideration for successful development of cell and growth factor-based regenerative strategies for the disc.

Topics: Animals; Cattle; Cells, Cultured; Extracellular Matrix; Hypoxia; Intervertebral Disc; Sepharose; Transforming Growth Factor beta3

Limited nutrient diffusion in three-dimensional (3D) constructs is a major concern in tissue engineering. Therefore, monitoring nutrient availability and diffusion within a scaffold is an important asset. Since nutrients come in various forms, we have investigated the diffusion of the oxygen, luciferin and dextran molecules within tissue-engineered constructs using optical imaging technologies. First, oxygen availability and diffusion were investigated, using transgenic cell lines in which a hypoxia-responsive element drives expression of the green fluorescent protein gene. Using confocal imaging, we observed oxygen limitation, starting at around 200 µm from the periphery in the context of agarose gel with 1 million CHO cells. Diffusion of luciferin was monitored real-time in agarose gels using a cell line in which the luciferase gene was driven by a constitutively active CMV promoter. Gel concentration affected the diffusion rate of luciferin. Furthermore, we assessed the diffusion rates of fluorescent dextran molecules of different molecular weights in biomaterials by fluorescence recovery after photobleaching (FRAP) and observed that diffusion depended on both molecular size and gel concentration. In conclusion, we have validated a set of efficient tools to investigate molecular diffusion of a range of molecules and to optimize biomaterials design in order to improve nutrient delivery.

Topics: Animals; Biocompatible Materials; Cell Survival; CHO Cells; Cricetinae; Cricetulus; Diffusion; Firefly Luciferin; Fluorescence Recovery After Photobleaching; Genes, Reporter; Green Fluorescent Proteins; Hypoxia; Imaging, Three-Dimensional; Luciferases; Microscopy, Confocal; Optics and Photonics; Oxygen; Sepharose; Tissue Engineering

Intracellular heat shock protein 72 (Hsp72) is known to serve a broad cytoprotective role. Recent data indicate that stressed cells can release Hsp72 into the extracellular compartment, although the biological function of extracellular Hsp72 remains to be fully elucidated. Because extracellular Hsp72 has been demonstrated to interact with Toll-like receptor 4, we hypothesized that endogenously produced and released Hsp72 would reprogram the mononuclear cell responses to LPS. THP-1 cells treated with LPS were used as a model for nuclear factor (NF)-kappaB activation. Heat shock conditions consisted of incubation at 43 degrees C for 1 h. Control cells were incubated at 37 degrees C. Twenty four hours after incubation, heat shock conditioned media (HSCM) and control media (CM) were centrifuged, and the respective cells were discarded. A separate group of naive THP-1 cells were then incubated with either HSCM or CM for 18 h and then stimulated with LPS (1 mug/mL). Heat shock significantly increased Hsp72 in HSCM compared with CM. In THP-1 cells transfected with an NF-kappaB luciferase reporter plasmid, the addition of HSCM attenuated subsequent LPS-mediated luciferase activity compared with cells incubated in CM. The addition of HSCM also attenuated LPS-mediated NF-kappaB-DNA binding and IkappaBalpha degradation. Heat shock protein 72-mediated inhibition of NF-kappaB activation was further corroborated by a significant decrease in TNF-alpha production. When HSCM and CM were subjected to Hsp72 depletion via adenosine triphosphate-agarose binding, LPS-mediated activation of NF-kappaB was partially restored, suggesting that Hsp72 is partially responsible for cellular reprogramming in response to HSCM. These data demonstrate that endogenously produced and released extracellular Hsp72 has the ability to reprogram the in vitro response to endotoxin in cultured human mononuclear cells.

Topics: Cell Line, Tumor; Endotoxins; HSP72 Heat-Shock Proteins; Humans; Hypoxia; Immune System; Inflammation; Ischemic Preconditioning; Leukocytes, Mononuclear; Lipopolysaccharides; Models, Biological; Monocytes; NF-kappa B; Sepharose; Signal Transduction

Our studies demonstrate that the ABC transporter and marker of stem and progenitor cells known as the breast cancer resistance protein (BCRP or ABCG2) confers a strong survival advantage under hypoxic conditions. We show that, under hypoxia, progenitor cells from Bcrp(-)/(-)mice have a reduced ability to form colonies as compared with progenitor cells from Bcrp(+/+) mice. Blocking BCRP function in Bcrp(+/+) progenitor cells markedly reduces survival under hypoxic conditions. However, blocking heme biosynthesis reverses the hypoxic susceptibility of Bcrp(-/-) progenitor cells, a finding that indicates that heme molecules (i.e. porphyrins) are detrimental to Bcrp(-/-) cells under hypoxia. BCRP specifically binds heme, and cells lacking BCRP accumulate porphyrins. Finally, Bcrp expression is up-regulated by hypoxia, and we demonstrate that this up-regulation involves the hypoxia-inducible transcription factor complex HIF-1. Collectively, our findings suggest that cells can, upon hypoxic demand, use BCRP to reduce heme or porphyrin accumulation, which can be detrimental to cells. Our findings have implications for the survival of stem cells and tumor cells in hypoxic environments.

Topics: Animals; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Biological Transport; Blotting, Western; Bone Marrow Cells; Cell Survival; Coloring Agents; Estrone; Heme; Hemin; Humans; Hypoxia; Mice; Mice, Transgenic; Models, Biological; Mutation; Neoplasm Proteins; NIH 3T3 Cells; Oxygen; Porphyrins; Promoter Regions, Genetic; Protein Binding; Protoporphyrins; Reverse Transcriptase Polymerase Chain Reaction; Sepharose; Signal Transduction; Stem Cells; Transcriptional Activation; Transgenes; Up-Regulation

Mitogen-activated protein kinase (MAPK) signaling cascades are multifunctional signaling networks that influence cell growth, differentiation, apoptosis, and cellular responses to stress. Since the activation/propagation of MAPK signaling requires the sequential phosphorylation of many downstream proteins, the phosphatases that dephosphorylate MAPKs represent critical elements in the control of MAPK-signaling networks. Here we show that hypoxia induces a transient increase in the activity of apoptosis signal-regulating kinase 1 (ASK-1), a MAPKKK that responds to oxidative stress by triggering cascades leading to the phosphorylation/activation of c-Jun N-terminal kinases (JNK) and p38-MAPK. Hypoxia-induced ASK-1/MKK-4/JNK signaling is suppressed by serine/threonine protein phosphatase type 5 (PP5), which acts to turn off ASK-1/MKK-4/JNK signaling via two mechanisms. First, in a rapid response hypoxia facilitates the association of endogenous PP5 with ASK-1. PP5 binds to the C-terminal domain of ASK-1, and studies with siRNA targeting PP5 indicate that PP5 acts to suppress the phosphorylation of MKK4 (Thr-261), JNK (Thr-183/Tyr-185), and c-Jun (Ser-63) without affecting the activating phosphorylation of p38 MAPK (Thr-180/Tyr-182), p44/p42-MAPK/ERK1/2 (Thr-202/Tyr-204), or c-Jun protein levels. If hypoxia is prolonged, the expression of PP5 is increased due to the activation of a transcriptional activator, which was identified as hypoxia-inducible factor-1. Together, these studies indicate that PP5 plays an important role in the survival of cells in a low oxygen environment by suppressing a hypoxia-induced ASK-1/MKK4/JNK signaling cascade that promotes an apoptotic response.

Topics: Apoptosis; Base Sequence; Blotting, Western; Cell Line; Cell Line, Tumor; Enzyme Activation; Genes, Reporter; Humans; Hypoxia; JNK Mitogen-Activated Protein Kinases; Luciferases; MAP Kinase Kinase 4; MAP Kinase Kinase Kinase 5; Microcystins; Mitogen-Activated Protein Kinase Kinases; Models, Biological; Molecular Sequence Data; Nuclear Proteins; Oxygen; Peptides, Cyclic; Phosphoprotein Phosphatases; Phosphorylation; Promoter Regions, Genetic; Protein Binding; Protein Structure, Tertiary; Reverse Transcriptase Polymerase Chain Reaction; RNA, Double-Stranded; RNA, Small Interfering; Sepharose; Sequence Homology, Nucleic Acid; Signal Transduction; Threonine; Time Factors; Transcriptional Activation

We have investigated inhibitory mechanisms of hypoxic activation of HIF-1alpha by nitric oxide (NO). Using a Hep3B cell-derived cell line, HRE7 cells, we found that the inhibition of HIF-1alpha activity by NO requires a substantial amount of oxygen, albeit at a lower level. We further investigated the effect of NO on the binding activity of the von Hippel-Lindau tumor suppressor protein (pVHL) to the N-terminal activation domain (NAD) overlapping the oxygen-dependent degradation domain (ODD) of HIF-1alpha, because this reaction involves prolyl hydroxylation in NAD that requires oxygen. Although we could not detect any binding activity when NAD was incubated with whole cell extracts from cells treated with CoCl(2) or desferrioxamine, the binding capacity was manifested when Hep3B cells were treated together with NO. This activation was also observed when whole cell extracts from CoCl(2)-treated cells were incubated with NO. The prolyl hydroxylase from Hep3B cells treated with CoCl(2) was partially purified about 80-fold, and several enzymatic properties were examined. The enzyme required ferrous ion and 2-oxoglutaric acid. Strong activation of the prolyl hydroxylase by NO was observed without further addition of ferrous ion.

Topics: Blotting, Western; Cell Line; Cobalt; DNA Fragmentation; DNA, Complementary; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Ketoglutaric Acids; Luciferases; Nitric Oxide; Oxygen; Procollagen-Proline Dioxygenase; Protein Binding; Protein Structure, Tertiary; Sepharose; Signal Transduction; Sodium Chloride; Time Factors; Transcription Factors; Transcription, Genetic; Transfection

Topics: Adenosine; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Animals; Blood Pressure; Coronary Disease; Disease Models, Animal; Hypoxanthines; Hypoxia; In Vitro Techniques; Inosine; Male; Microspheres; Myocardial Contraction; Myocardium; Perfusion; Phosphocreatine; Polysaccharides; Rats; Sepharose