carbocyanines and Hypoxia

Photocaging holds promise for the precise manipulation of biological events in space and time. However, current near-infrared (NIR) photocages are oxygen-dependent for their photolysis and lack of timely feedback regulation, which has proven to be the major bottleneck for targeted therapy. Herein, we present a hypoxia-dependent photo-activation mechanism of dialkylamine-substituted cyanine (Cy-NH) accompanied by emissive fragments generation, which was validated with retrosynthesis and spectral analysis. For the first time, we have realized the orthogonal manipulation of this hypoxia-dependent photocaging and dual-modal optical signals in living cells and tumor-bearing mice, making a breakthrough in the direct spatiotemporal control and in vivo feedback regulation. This unique photoactivation mechanism overcomes the limitation of hypoxia, which allows site-specific remote control for targeted therapy, and expands the photo-trigger toolbox for on-demand drug release, especially in a physiological context with dual-mode optical imaging under hypoxia.

Topics: A549 Cells; Animals; Carbocyanines; Drug Liberation; Hep G2 Cells; Humans; Hypoxia; Infrared Rays; Mice; Molecular Structure; Neoplasms, Experimental; Optical Imaging; Photoacoustic Techniques; Photolysis

Programmable DNA-based nanostructures (

Topics: Animals; Carbocyanines; DNA; Fluorescent Dyes; Humans; Hypoxia; Light; MCF-7 Cells; Mice; Mitochondria; Nanostructures; Neoplasms; Nitroreductases; Photochemotherapy; Photosensitizing Agents; Porphyrins; Singlet Oxygen

Hypoxia is associated with clinical diseases. Extreme hypoxia leads to multiple organs failure. However, the different effects of hypoxia on brain and visceral organs still need to be clarified, and moreover, characteristics in vulnerable organs suffering from hypoxia remain elusive. In the present study, we first aimed to figure out the hypoxic sensitivity of organs. Adult male mice were exposed to 6% O

Topics: Animals; Behavior, Animal; Blotting, Western; Brain; Carbocyanines; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney; Liver; Magnetic Resonance Spectroscopy; Male; Mice, Inbred BALB C; Molecular Imaging; Nanocomposites

Mitochondria, powerhouses of cells, possess a weakly alkaline environment. Various stress stimulations may lead to mitophagy, which further gives a rise to mitochondrial acidification and disfunction. Therefore, monitoring mitochondrial pH alterations is of great importance to better elucidate their role in the cellular metabolism. Toward this end, a number of mitochondrial fluorescent pH probes have been proposed, but most of them are based on electrostatic attraction and readily leak out from the mitochondria during mitophagy with decreased membrane potential, thus failing to accurately measure the pH changes. In this work, we report a mitochondria-immobilized ratiometric fluorescent pH probe, which allows the quantitative measurements of mitochondrial pH. The probe was designed and prepared by introducing a reactive benzyl chloride into a positively charged near-infrared hydroxyl-hemicyanine. The cationic property facilitates the probe to be quickly enriched into mitochondria, the hydroxyl group is responsible for producing a reversible ratiometric fluorescence signal, and benzyl chloride is used to react with nucleophiles for immobilizing the probe in mitochondria. Taking these advantages of the probe, the mitochondrial pH variations during mitophagy caused by rapamycin and hypoxia have been determined quantitatively for the first time. The observed severe acidification of mitochondria under these stimulations, together with the rationally designed probe, may be useful for studying the detailed function of mitochondria in some bioprocesses.

Topics: Benzyl Compounds; Carbocyanines; Fluorescent Dyes; HeLa Cells; Humans; Hydrogen-Ion Concentration; Hypoxia; Mitochondria; Mitophagy; Sirolimus

Pulmonary arterial hypertension (PAH) is characterized by enhanced proliferation of pulmonary artery smooth muscle cells and endothelial cells associated with obliteration of small pulmonary arterioles and formation of plexiform lesions. To date, no curative treatments have been identified for pulmonary arterial hypertension. There are various therapeutic options, including conventional medical therapies and oral, subcutaneous, intravenous, and inhalation delivery. We have previously shown that miR-143/145 knockout can prevent the development of chronic hypoxia-induced pulmonary hypertension (PH) in mice. Here, we use chronic hypoxia-induced PH as a disease model to evaluate miR-143/145 inhibition after delivery of antimiRNAs via the subcutaneous or intranasal routes. We use qRT-PCR and immunofluorescence to confirm that both delivery strategies efficiently inhibit miR-143/145 in lung tissue from mice with chronic hypoxia-induced PH.

Topics: Administration, Intranasal; Animals; Antagomirs; Carbocyanines; Chronic Disease; Disease Models, Animal; Female; Fluorescent Antibody Technique; Gene Transfer Techniques; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Lung; Mice, Inbred C57BL; MicroRNAs; Real-Time Polymerase Chain Reaction

Nitroreductase (NTR) can be overexpressed in hypoxic tumors, thus the selective and efficient detection of NTR is of great importance. To date, although a few optical methods have been reported for the detection of NTR in solution, an effective optical probe for NTR monitoring in vivo is still lacking. Therefore, it is necessary to develop a near-infrared (NIR) fluorescent detection probe for NTR. In this study, five NIR cyanine dyes with fluorescence reporting structure decorated with different nitro aromatic groups, Cy7-1-5, have been designed and explored for possible rapid detection of NTR. Our experimental results presented that only a para-nitro benzoate group modified cyanine probe (Cy7-1) could serve as a rapid NIR fluorescence-enhanced probe for monitoring and bioimaging of NTR. The structure-function relationship has been revealed by theoretical study. The linker connecting the detecting and fluorescence reporting groups and the nitro group position is a key factor for the formation of hydrogen bonds and spatial structure match, inducing the NTR catalytic ability enhancement. The in vitro response and mechanism of the enzyme-catalyzed reduction of Cy7-1 have been investigated through kinetic optical studies and other methods. The results have indicated that an electro-withdrawing group induced electron-transfer process becomes blocked when Cy7-1 is catalytically reduced to Cy7-NH2 by NTR, which is manifested in enhanced fluorescence intensity during the detection process. Confocal fluorescence imaging of hypoxic A549 cells has confirmed the NTR detection ability of Cy7-1 at the cellular level. Importantly, Cy7-1 can detect tumor hypoxia in a murine hypoxic tumor model, showing a rapid and significant enhancement of its NIR fluorescence characteristics suitable for fluorescence bioimaging. This method may potentially be used for tumor hypoxia diagnosis.

Topics: Animals; Benzothiazoles; Carbocyanines; Cell Line, Tumor; Fluorescence; Fluorescent Dyes; Humans; Hypoxia; Mice; Microscopy, Confocal; Microscopy, Fluorescence; Molecular Docking Simulation; Neoplasms; Nitroreductases; Optical Imaging; Whole Body Imaging

Brain tumors and brain metastases are among the deadliest malignancies of all human cancers, largely due to the cellular blood-brain and blood-tumor barriers that limit the delivery of imaging and therapeutic agents from the systemic circulation to tumors. Thus, improved strategies for brain tumor visualization and targeted treatment are critically needed. Here we identified and synthesized a group of near-infrared fluorescence (NIRF) heptamethine carbocyanine dyes and derivative NIRF dye-drug conjugates for effective imaging and therapeutic targeting of brain tumors of either primary or metastatic origin in mice, which is mechanistically mediated by tumor hypoxia and organic anion-transporting polypeptide genes. We also demonstrate that these dyes, when conjugated to chemotherapeutic agents such as gemcitabine, significantly restricted the growth of both intracranial glioma xenografts and prostate tumor brain metastases and prolonged survival in mice. These results show promise in the application of NIRF dyes as novel theranostic agents for the detection and treatment of brain tumors.

Topics: Animals; Blood-Brain Barrier; Brain Neoplasms; Carbocyanines; Cell Line, Tumor; Deoxycytidine; Diagnostic Imaging; Drug Delivery Systems; Fluorescent Dyes; Gemcitabine; HEK293 Cells; Humans; Hypoxia; Male; Mice, Nude; Mice, SCID; Neoplasm Metastasis; Organic Anion Transporters; Prostatic Neoplasms; Spectroscopy, Near-Infrared; Xenograft Model Antitumor Assays

Alterations of mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and mitochondrial respiration are possible triggers of pulmonary vascular remodeling in pulmonary hypertension (PH). We investigated the role of MMP in PH and hypothesized that deletion of the mitochondrial uncoupling protein 2 (UCP2) increases MMP, thus promoting pulmonary vascular remodeling and PH. MMP was measured by JC-1 in isolated pulmonary arterial smooth muscle cells (PASMCs) of patients with PH and animals with PH induced by exposure to monocrotaline (MCT) or chronic hypoxia. PH was quantified in vivo in UCP2-deficient (UCP2(-/-)) mice by hemodynamics, morphometry, and echocardiography. ROS were measured by electron spin resonance spectroscopy and proliferation by thymidine incorporation. Mitochondrial respiration was investigated by high-resolution respirometry. MMP was increased in PASMCs of patients and in animal models of PH. UCP2(-/-) mice exhibited pulmonary vascular remodeling and mild PH compared with wild-type (WT) mice. PASMCs of UCP2(-/-) mice showed increased proliferation, MMP, and ROS release. Increased proliferation of UCP2(-/-) PASMCs could be attenuated by ROS inhibitors and inhibited by carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, which decreased MMP to the level of WT mice. Mitochondrial respiration was altered in PASMCs from MCT rats and PASMCs exposed to hypoxia but not in isolated pulmonary mitochondria of UCP2(-/-) mice or PASMCs after treatment with small interfering RNA for UCP2. Our data suggest that increased MMP causes vascular remodeling in UCP2(-/-) mice partially via increased ROS. In chronic hypoxia and MCT-induced PH, additional pathomechanisms such as decreased respiration may play a role.

Topics: Animals; Benzimidazoles; Carbocyanines; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Disease Models, Animal; Fluorescent Dyes; Free Radical Scavengers; Gene Expression Regulation; Humans; Hypertension, Pulmonary; Hypoxia; Ion Channels; Membrane Potential, Mitochondrial; Mice; Mitochondria; Mitochondrial Proteins; Monocrotaline; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Primary Cell Culture; Pulmonary Artery; Rats; Reactive Oxygen Species; RNA, Small Interfering; Uncoupling Protein 2

Ischemic tolerance is an endogenous neuroprotective mechanism in brain and other organs, whereby prior exposure to brief ischemia produces resilience to subsequent normally injurious ischemia. Although many molecular mechanisms mediate delayed (gene-mediated) ischemic tolerance, the mechanisms underlying rapid (protein synthesis-independent) ischemic tolerance are relatively unknown. Here we describe a novel mechanism for the induction of rapid ischemic tolerance mediated by the ubiquitin-proteasome system. Rapid ischemic tolerance is blocked by multiple proteasome inhibitors [carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG132), MG115 (carbobenzoxy-L-leucyl-L-leucyl-L-norvalinal), and clasto-lactacystin-beta-lactone]. A proteomics strategy was used to identify ubiquitinated proteins after preconditioning ischemia. We focused our studies on two actin-binding proteins of the postsynaptic density that were ubiquitinated after rapid preconditioning: myristoylated, alanine-rich C-kinase substrate (MARCKS) and fascin. Immunoblots confirm the degradation of MARCKS and fascin after preconditioning ischemia. The loss of actin-binding proteins promoted actin reorganization in the postsynaptic density and transient retraction of dendritic spines. This rapid and reversible synaptic remodeling reduced NMDA-mediated electrophysiological responses and renders the cells refractory to NMDA receptor-mediated toxicity. The dendritic spine retraction and NMDA neuroprotection after preconditioning ischemia are blocked by actin stabilization with jasplakinolide, as well as proteasome inhibition with MG132. Together these data suggest that rapid tolerance results from changes to the postsynaptic density mediated by the ubiquitin-proteasome system, rendering neurons resistant to excitotoxicity.

Topics: Analysis of Variance; Animals; Animals, Newborn; Carbocyanines; Carrier Proteins; Cell Death; Cells, Cultured; Cerebral Cortex; Enzyme Inhibitors; Glucose; Hypoxia; Intracellular Signaling Peptides and Proteins; Ischemia; Ischemic Preconditioning; Membrane Potentials; Membrane Proteins; Microfilament Proteins; Myristoylated Alanine-Rich C Kinase Substrate; Neurons; Patch-Clamp Techniques; Proteasome Endopeptidase Complex; Rats; Rats, Sprague-Dawley; Synapses; Time Factors; Ubiquitin

Maternal endothelial activation in pre-eclampsia is attributed to the release of unknown factors from a hypoperfused placenta. To further characterize these factors, we have used a serum-free placental villous explant culture model and investigated the effect of the liberated soluble factors produced on human endothelial cell cultures. Term placental villous explants from uncomplicated pregnancies were cultured for 4 days in 20, 6 or 1% O2 to mimic placental hyperoxia, normoxia and hypoxia. Medium collected from viable explants was applied to cultured human uterine microvascular endothelial cells. Medium conditioned by hypoxic explants caused a significant decrease in endothelial cell ATP levels and mitochondrial dehydrogenase activity, suggestive of a reduced metabolic rate. An additional reduction in mitochondrial membrane potential and increased endothelial cell death occurred as the oxygen concentration to which explants had been exposed decreased. Effects of the hypoxic explant medium were also seen ex vivo in a wire myography model of myometrial artery function, with increased vasoconstriction and attenuated vasodilation following exposure to hypoxic explant medium. These results suggest that hypoxia (1% O2) may stimulate the release of soluble factors from the placenta, which have an adverse effect on endothelial cell metabolism and mitochondrial integrity in vitro. These potentially pathogenic factors are now being characterized.

Topics: Apoptosis; Arginine Vasopressin; Benzimidazoles; Bradykinin; Carbocyanines; Cells, Cultured; Chorionic Villi; Dose-Response Relationship, Drug; Endothelin-1; Endothelium, Vascular; Epoprostenol; Female; Formazans; Humans; Hyperoxia; Hypoxia; Membrane Potentials; Mitochondria; Myometrium; Necrosis; Neovascularization, Physiologic; Oxygen; Placenta; Pregnancy; Tetrazolium Salts; Vasodilator Agents

Developing oligodendrocytes (OL precursors, pre-OLs) express alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) subtype glutamate receptors (AMPARs) and are highly vulnerable to hypoxic-ischemic or oxygen-glucose deprivation (OGD)-induced excitotoxic injury, yet the mechanisms of injury remain unclear. Here we investigated the role of glutamate accumulation and mitochondrial function in OGD-induced pre-OL toxicity in vitro. Bulk glutamate concentration in the culture medium did not increase during OGD and OGD-conditioned medium did not transfer toxicity to naïve cells. Facilitation of glutamate diffusion by constant agitation of the culture reduced, while inhibition of glutamate diffusion by increasing medium viscosity with dextran enhanced, OGD-induced pre-OL injury. Depletion of extracellular glutamate by the glutamate scavenging system, glutamate-pyruvate transaminase plus pyruvate, attenuated pre-OL injury during OGD. Together these data suggest that local glutamate accumulation is critical for OGD toxicity. Interestingly, under normoxic conditions, addition of glutamate to pre-OLs did not cause receptor-mediated toxicity, but the toxicity could be unmasked by mitochondrial impairment with mitochondrial toxins. Furthermore, OGD caused mitochondrial potential collapse that was independent of AMPAR activation, and OGD toxicity was enhanced by mitochondrial toxins. These data demonstrate that pre-OL excitotoxicity is exacerbated by mitochondrial dysfunction during OGD. Overall, our results indicate that OGD-induced pre-OL injury is a novel form of excitotoxicity caused by the combination of local glutamate accumulation that occurs without an increase in bulk glutamate concentration and mitochondrial dysfunction. Therapeutic strategies targeting local glutamate concentration and mitochondrial injury during hypoxia-ischemia may be relevant to human disorders associated with pre-OL excitotoxicity.

Topics: Animals; Animals, Newborn; Benzimidazoles; Brain; Calcium; Carbocyanines; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Drug Interactions; Excitatory Amino Acid Antagonists; Glucose; Glutamic Acid; Hypoxia; In Situ Nick-End Labeling; Ionophores; Mitochondria; Oligodendroglia; Quinoxalines; Rats; Rats, Sprague-Dawley; Time Factors

Proximal tubules develop a severe energetic deficit during hypoxia-reoxygenation (H/R) that previous studies using fluorescent potentiometric probes have suggested is characterized by sustained, partial mitochondrial deenergization. To validate the primary occurrence of mitochondrial deenergization in the process, optimize approaches for estimating changes in mitochondrial membrane potential (DeltaPsim) in the system, and clarify the mechanisms for the defect, we further investigated the behavior of 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazocarbocyanine iodide (JC-1) in these cells and introduce a more dynamic and quantitative approach employing safranin O for use with the tubule system. Although use of JC-1 can be complicated by decreases in the plasma membrane potential that limit cellular uptake of JC-1 and such behavior was demonstrated in ouabain-treated tubules, changes in DeltaPsim entirely accounted for the decreases in the formation of red fluorescent JC-1 aggregates and in the ratio of red/green fluorescence observed after H/R. The red JC-1 aggregates did not readily dissociate when tubules were deenergized after JC-1 uptake, making it unsuitable for dynamic studies of energization. Safranin O uptake by digitonin-permeabilized tubules required very small numbers of tubules, permitted measurements of DeltaPsim for relatively prolonged periods after the end of the experimental maneuvers, was rapidly reversible during deenergization, and allowed for direct assessment of both substrate-dependent, electron transport-mediated DeltaPsim, and ATP hydrolysis-supported DeltaPsim. Both types of energization measured using safranin O in tubules permeabilized after H/R were impaired, but combining substrates and ATP substantially restored DeltaPsim.

Topics: Acute Kidney Injury; Adenosine Triphosphate; Animals; Benzimidazoles; Carbocyanines; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Membrane Permeability; Coloring Agents; Energy Metabolism; Enzyme Inhibitors; Female; Fluorescent Dyes; Hypoxia; Ionophores; Kidney Tubules, Proximal; Membrane Potentials; Mitochondria; Ouabain; Phenazines; Proton-Translocating ATPases; Rabbits

Mitochondrial membrane potential (MMP) regulates the production of high-energy phosphate and apoptotic cascade, both occurring after ischemic impact. The timed profile of MMP differing from grading ischemic impact has to be determined. Primary rat hippocampal cultures were exposed to oxygen-glucose deprivation (OGD) for 30, 60, and 90 min and then were reoxygenated. MMP was expressed as a voltage-dependent dye, JC-1 fluorescence, under confocal microscopy. Cell viability was assessed by calcein AM and ethidium homodimer, each at 3 hours and 24 hours after 30, 60, and 90 min of OGD. The appearance of apoptosis was also evaluated by the TUNEL method at 24 hours. Hyperpolarization of MMP (2.31+/-0.94 normalized JC-1 fluorescence ratio between red and green) was observed during reoxygenation after 30 min OGD, while 60 min OGD induced depolarization (0.66+/-0.22, Valinomycin (potassium ionophore)-induced depolarization: 0.53+/-0.19). The fluorescence of mitochondria became weak after 90 min OGD. Most of the neurons were shrunken after 90 min and neurons were TUNEL-positive 24 hours after 30 min OGD, although most neurons were viable at 3 hours. A longer period of OGD induced necrosis, and most neurons remained viable after only 3 hours. Our data present that the short (30 min) OGD induced hyperpolarization of MMP during reoxygenation, while a longer OGD (60 or 90 min) induced depolarization and acute necrosis. Neurons were still viable even during hyperpolarization of mitochondria, but this hyperpolarization appears to be linked to subsequent apoptotic change.

Topics: Animals; Animals, Newborn; Antigens; Apoptosis; Benzimidazoles; Bromodeoxyuridine; Carbocyanines; CD11b Antigen; Cell Death; Cell Survival; Cells, Cultured; Fluoresceins; Galactosylceramides; Glial Fibrillary Acidic Protein; Glucose; Hippocampus; Humans; Hypoxia; In Situ Nick-End Labeling; Membrane Potentials; Mice; Microtubule-Associated Proteins; Mitochondria; Neurons; Oligodendroglia; Oxygen; Rats; Time Factors; von Willebrand Factor

We have reinvestigated the hypothesis of the relative importance of glomus cell plasma and mitochondrial membrane potentials (E(m) and psi(m), respectively) in acute hypoxia by a noninvasive fluorescence microimaging technique using the voltage-sensitive dyes bis-oxonol and JC-1, respectively. Short-term (24 h)-cultured rat glomus cells and cultured PC-12 cells were used for the study. Glomus cell E(m) depolarization was indirectly confirmed by an increase in bis-oxonol (an anionic probe) fluorescence due to a graded increase in extracellular K(+). Fluorescence responses of glomus cell E(m) to acute hypoxia (approximately 10 Torr Po(2)) indicated depolarization in 20%, no response in 45%, and hyperpolarization in 35% of the cells tested, whereas all PC-12 cells consistently depolarized in response to hypoxia. Furthermore, glomus cell E(m) hyperpolarization was confirmed with high CO (approximately 500 Torr). Glomus cell psi(m) depolarization was indirectly assessed by a decrease in JC-1 (a cationic probe) fluorescence. Accordingly, 1 microM carbonyl cyanide p-trifluoromethoxyphenylhydrazone (an uncoupler of oxidative phosphorylation), high CO (a metabolic inhibitor), and acute hypoxia (approximately 10 Torr Po(2)) consistently depolarized the mitochondria in all glomus cells tested. Likewise, all PC-12 cell mitochondria depolarized in response to FCCP and hypoxia. Thus, although bis-oxonol could not show glomus cell depolarization consistently, JC-1 monitored glomus cell mitochondrial depolarization as an inevitable phenomenon in hypoxia. Overall, these responses supported our "metabomembrane hypothesis" of chemoreception.

Topics: Acute Disease; Animals; Benzimidazoles; Carbocyanines; Carbon Monoxide; Carotid Body; Fluorescent Dyes; Hypoxia; Membrane Potentials; Microscopy, Fluorescence; Mitochondria; Oxygen; Patch-Clamp Techniques; PC12 Cells; Potassium; Rats; Rats, Sprague-Dawley; Thiobarbiturates

Kidney proximal tubule cells developed severe energy deficits during hypoxia/reoxygenation not attributable to cellular disruption, lack of purine precursors, the mitochondrial permeability transition, or loss of cytochrome c. Reoxygenated cells showed decreased respiration with complex I substrates, but minimal or no impairment with electron donors at complexes II and IV. This was accompanied by diminished mitochondrial membrane potential (DeltaPsi(m)). The energy deficit, respiratory inhibition, and loss of DeltaPsi(m) were strongly ameliorated by provision of alpha-ketoglutarate plus aspartate (alphaKG/ASP) supplements during either hypoxia or only during reoxygenation. Measurements of (13)C-labeled metabolites in [3-(13)C]aspartate-treated cells indicated the operation of anaerobic pathways of alphaKG/ASP metabolism to generate ATP, yielding succinate as end product. Anaerobic metabolism of alphaKG/ASP also mitigated the loss of DeltaPsi(m) that occurred during hypoxia before reoxygenation. Rotenone, but not antimycin or oligomycin, prevented this effect, indicating that electron transport in complex I, rather than F(1)F(0)-ATPase activity, had been responsible for maintenance of DeltaPsi(m) by the substrates. Thus, tubule cells subjected to hypoxia/reoxygenation can have persistent energy deficits associated with complex I dysfunction for substantial periods of time before onset of the mitochondrial permeability transition and/or loss of cytochrome c. The lesion can be prevented or reversed by citric acid cycle metabolites that anaerobically generate ATP by intramitochondrial substrate-level phosphorylation and maintain DeltaPsi(m) via electron transport in complex I. Utilization of these anaerobic pathways of mitochondrial energy metabolism known to be present in other mammalian tissues may provide strategies to limit mitochondrial dysfunction and allow cellular repair before the onset of irreversible injury by ischemia or hypoxia.

Topics: Adenosine Triphosphate; Animals; Aspartic Acid; Benzimidazoles; Carbocyanines; Citric Acid Cycle; Fluorescent Dyes; Hypoxia; Ketoglutaric Acids; Kidney Tubules; Membrane Potentials; Mitochondria; Models, Biological; Oxidative Phosphorylation; Oxygen; Rabbits; Time Factors

Episodes of tissue hypoxia and reoxygenation frequently occur during gram-negative bacteremia that progresses to septic shock. However, few studies have evaluated modulation by hypoxia and reoxygenation of the proinflammatory cytokine gene expression that is normally induced by gram-negative bacteremia or endotoxemia. In buffer-perfused organs, hypoxia downregulates Escherichia coli-induced expression of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta in the liver but upregulates these cytokines in the lungs. To identify molecular mechanisms underlying these events, we investigated the effects of brief (1.5-h) hypoxia on TNF-alpha and IL-1beta expression in cultured RAW 264.7 cells during their continuous exposure to lipopolysaccharide (LPS) endotoxin derived from E. coli (serotype 055:B5) for up to 24 h. IL-1beta and TNF-alpha concentrations in cell lysates and culture supernatants were measured by ELISA, and steady-state mRNA was measured by Northern analysis. LPS-induced IL-1beta synthesis was downregulated by hypoxia at both the protein and mRNA levels despite no change in cellular redox status as measured by levels of GSH. In contrast, LPS-induced TNF-alpha production was unaffected by hypoxia as assessed by cell lysate mRNA and lysate and supernatant protein levels. Nuclear runoff analysis showed that downregulation of IL-1beta gene expression by hypoxia occurred transcriptionally. Allopurinol or catalase treatment did not alter modulation of LPS-induced IL-1beta expression by hypoxia, suggesting that this suppression was not caused by reactive oxygen species. Cycloheximide pretreatment suggested that hypoxia-induced downregulation of IL-1beta expression did not require de novo protein synthesis.

Topics: Animals; Carbocyanines; Catalase; Cell Line; Homeostasis; Hypoxia; Interleukin-1; Lipopolysaccharides; Mice; RNA, Messenger; Transcription, Genetic; Tumor Necrosis Factor-alpha

Angiogenesis and coronary artery collateral formation can improve blood flow and thereby prevent myocardial ischemia. The role of perivascular fibroblasts in neovascularization remains incompletely understood. Here we investigated the effects of epicardial and myocardial fibroblasts on angiogenesis in vitro by using a serum-free microcarrier-based fibrin gel angiogenesis system. To clearly distinguish between different cell types, we either stained endothelial cells or fibroblasts in the living with 1, 1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine-perchlorate (DiI). In cocultures, low numbers of heart fibroblasts stimulated endothelial sprouting, and capillary growth was also induced by fibroblast-conditioned media, indicating a paracrine mechanism. Capillary formation was decreased by increasing the density of fibroblasts in the cocultures, indicating contact-dependent inhibition. Using time-lapse studies, it turned out that close contacts between fibroblasts and endothelial cells resulted in rapid retraction of endothelial cells or, rarely, in cell death. Depending on the local ratio of fibroblasts to endothelial cell numbers, fibroblasts determined the location of capillary growth and the size of developing capillaries and thereby contributed to capillary network remodeling. In contrast to primary heart fibroblasts, NIH 3T3 fibroblasts did not display contact-dependent inhibition of endothelial sprouts. NIH fibroblasts were frequently seen in close association with endothelial capillaries, resembling pericytes. Contact-dependent inhibition of angiogenesis by epicardial fibroblasts could not be reversed by addition of neutralizing anti-TGF-beta1 antibodies, by addition of serum, of medium conditioned by hypoxic tumor cells or myocardium, by various cytokines or by growing cocultures under hypoxic conditions. Our results implicate a pivotal role of periendothelial mesenchymal cells for the regulation of microvascular network remodeling and collateral formation.

Topics: Animals; Carbocyanines; Cell Communication; Coculture Techniques; Collateral Circulation; Coronary Circulation; Coronary Vessels; Culture Media, Conditioned; Culture Media, Serum-Free; Endothelium, Vascular; Fibrin; Fibroblasts; Gels; Humans; Hypoxia; Myocardium; Neovascularization, Physiologic; Swine

Selective degeneration of postsynaptic neuronal dendrites is a pathological hallmark of brain injury in stroke and other neurological disorders. We examined dendritic injury in primary cultures dissociated from mouse neocortex. Neuronal morphology was visualized using the fluorescent membrane tracer, Dil, or immunofluorescence with antibodies to the dendrite-specific microtubule-associated protein, MAP2. Deprivation of oxygen and glucose for 30-60 min resulted in segmental dendritic beading, or varicosities, and loss of dendritic spines. This pattern of dendritic injury was blocked by addition of selective NMDA antagonists, and was reproduced within 5 min of exposure to 10-100 microM NMDA. Widespread dendritic varicosity formation occurred even with exposures to oxygen-glucose deprivation or NMDA which resulted in little neuronal death by the following day. Despite marked structural changes affecting virtually all neurons, dendrite shape returned to normal within 2 h of terminating sublethal oxygen-glucose deprivation or NMDA application. Rapid, reversible changes in dendritic structure may contribute to alterations in neuronal function following glutamate receptor stimulation under physiological or pathological conditions.

Topics: Animals; Carbocyanines; Cell Death; Cerebral Cortex; Dendrites; Fluorescent Antibody Technique; Fluorescent Dyes; Glucose; Hypoxia; Mice; Microtubule-Associated Proteins; Neurons; Receptors, Glutamate; Receptors, N-Methyl-D-Aspartate

Topics: Animals; Benzimidazoles; Blood Flow Velocity; Carbocyanines; Fluorescent Dyes; Hypoxia; Mice; Mice, Inbred C3H; Oxygen Consumption; Oxyhemoglobins; Perfusion; Sarcoma, Experimental; Tumor Stem Cell Assay