Kosuke Tsukada

Perivascular nitric oxide gradients normalize tumor vasculature

Normalization of tumor vasculature is an emerging strategy to improve cytotoxic therapies. Here we show that eliminating nitric oxide (NO) production from tumor cells via neuronal NO synthase silencing or inhibition establishes perivascular gradients of NO in human glioma xenografts in mice and normalizes the tumor vasculature, resulting in improved tumor oxygenation and response to radiation treatment. Creation of perivascular NO gradients may be an effective strategy for normalizing abnormal vasculature.

Roles of Hemoglobin Allostery in Hypoxia-induced Metabolic Alterations in Erythrocytes SIMULATION AND ITS VERIFICATION BY METABOLOME ANALYSIS

When erythrocytes are exposed to hypoxia, hemoglobin (Hb) stabilizes in the T-state by capturing 2,3-bisphosphoglycerate. This process could reduce the intracellular pool of glycolytic substrates, jeopardizing cellular energetics. Recent observations suggest that hypoxia-induced activation of glycolytic enzymes is correlated with their release from Band III (BIII) on the cell membrane. Based on these data, we developed a mathematical model of erythrocyte metabolism and compared hypoxia-induced differences in predicted activities of the enzymes, their products, and cellular energetics between models with and without the interaction of Hb with BIII. The models predicted that the allostery-dependent Hb interaction with BIII accelerates consumption of upstream glycolytic substrates such as glucose 6-phosphate and increases downstream products such as phosphoenolpyruvate. This prediction was consistent with metabolomic data from capillary electrophoresis mass spectrometry. The hypoxia-induced alterations in the metabolites resulted from acceleration of glycolysis, as judged by increased conversion of [13C]glucose to [13C]lactate. The allostery-dependent interaction of Hb with BIII appeared to contribute not only to maintenance of energy charge but also to further synthesis of 2,3-bisphosphoglycerate, which could help sustain stabilization of T-state Hb during hypoxia. Furthermore, such an activation of glycolysis was not observed when Hb was stabilized in R-state by treating the cells with CO. These results suggest that Hb allostery in erythrocytes serves as an O2-sensing trigger that drives glycolytic acceleration to stabilize intracellular energetics and promote the ability to release O2 from the cells.

Development of catheter-type optical oxygen sensor and applications to bioinstrumentation

A catheter-type optical oxygen sensor based on phosphorescence lifetime was developed for medical and animal experimental use. Since the sensor probe should have biocompatibility and high oxygen permeability in vivo, we focused attention on acceptable polymer materials for contact lenses as the substrates of probes. Pd-porphyrin was doped in silicone-based polymer, and was fixed at the edge of an optical fiber inserted in a catheter tube. The shape of the probe was 600 microm in diameter and 100 microm in thickness, and the probe had high oxygen permeability of Dk value 455. In accuracy evaluation, there found an excellent correlation between the pO2 values measured through phosphorescence lifetime using the oxygen sensors and those measured as the calibrating data using oxygen electrodes. The response time required to achieve 90% from reversible default value to be from 150 to 0 mmHg, and from 0 to 150 mmHg was 15.43 and 7.52 s, respectively. In addition, other properties such as temperature and pH dependency, response, and durability of our optical oxygen sensor were investigated. In animal experiments, the catheter-type oxygen sensor was inserted via the femoral artery of a rat, and arterial oxygen pressure was monitored under asphyxiation. The sensor was valid in the range of oxygen concentration sufficient for biometry, and expected to be integrated with an indwelling needle.

Near-infrared laser adjuvant for influenza vaccine.

Safe and effective immunologic adjuvants are often essential for vaccines. However, the choice of adjuvant for licensed vaccines is limited, especially for those that are administered intradermally. We show that non-tissue damaging, near-infrared (NIR) laser light given in short exposures to small areas of skin, without the use of additional chemical or biological agents, significantly increases immune responses to intradermal influenza vaccination without augmenting IgE. The NIR laser-adjuvanted vaccine confers increased protection in a murine influenza lethal challenge model as compared to unadjuvanted vaccine. We show that NIR laser treatment induces the expression of specific chemokines in the skin resulting in recruitment and activation of dendritic cells and is safe to use in both mice and humans. The NIR laser adjuvant technology provides a novel, safe, low-cost, simple-to-use, potentially broadly applicable and clinically feasible approach to enhancing vaccine efficacy as an alternative to chemical and biological adjuvants.

Platelet adhesion and arteriolar dilation in the photothrombosis: observation with the rat closed cranial and spinal windows.

The mechanism of cerebral infarction, in which thrombus formation and platelet-endothelium interaction play an important part, have not yet been clearly elucidated in vivo. The aim of this study was to observe rolling and adherent platelets and to analyze adherent leukocytes and vessel diameter change in vivo using a photothrombotic vessel occlusion model.A photothrombosis, which is mediated by free radicals, was induced in male Wistar rats in the presence of a photosensitizing dye (Photofrin II) and exposure to a filtered light. Rhodamine 6G-labeled platelets and leukocytes were visualized with intravital fluorescence videomicroscopy through a closed cranial or spinal window. The vessel diameter, photothrombosis and leukocyte adhesion were analyzed. Rolling and adherent platelets were observed during irradiation through the cerebral and spinal window. Before the platelets were recognized, the irradiated arteriole dilated significantly. After the photochemical occlusion of an arteriole, other arterioles also dilated and the adherent leukocytes increased in the venules. The photothrombosis were almost completely composed of platelets according to electron microscopic analysis. The arteriolar dilation rate and the number of adherent leukocytes in the cerebrum were greater than those in the spinal cord. By combining the photochemical thrombus formation and the fluorescence microscope techniques, we were able for the first time to observe rolling and adherent platelets and microvascular responses during photothrombosis in the cerebral and spinal microvasculature. It is suggested that free radicals, which can lead to platelet aggregation, play an important role as a cerebral vasodilator. This model is useful for cerebral and spinal microcirculatory analysis to investigate the platelet-endothelium interaction, the platelet aggregation and the effect of free radicals on cerebral and spinal microcirculation.

An in vitro hepatic zonation model with a continuous oxygen gradient in a microdevice.

In a hepatic lobule, different sets of metabolic enzymes are expressed in the periportal (PP) and pericentral (PC) regions, forming a functional zonation, and the oxygen gradient is considered a determinant of zone formation. It is desirable to reproduce lobular microenvironment in vitro, but incubation of primary hepatocytes in conventional culture dishes has been limited at fixed oxygen concentrations due to technical difficulties. We designed a cell culture microdevice with an oxygen gradient to reproduce the hepatic microenvironment in vitro. The oxygen gradient during cell culture was monitored using a laser-assisted phosphorescence quenching method, and the cellular oxygen consumption rate could be estimated from changes in the gradient. Culture medium was continuously exchanged through microchannels installed in the device to maintain the oxygen gradient for a long term without transient hyper-oxygenation. The oxygen consumption rates of hepatocytes at 70.0mmHg and 31.4mmHg of partial oxygen pressure, which correspond to PP and PC regions in the microdevice, were 3.67×10(-10) and 3.15×10(-10)mol/s/10(6) cells, respectively. Antimycin A changed the oxygen gradient profile, indicating that cellular respiration can be estimated during cell culture. RT-PCR analysis of hepatocytes cultured under the oxygen gradient showed that mRNA expression of PEPCK and GK significantly increased in culture areas corresponding to PP and PC regions, respectively. These results indicate that the developed microdevice can reproduce the hepatic lobular microenvironment. The oxygen gradient in the microdevice can be closely controlled by changing the sizes of gas channels and the ambient oxygen concentration around the device; therefore, it could be expected to mimic the oxygen gradient of various organs, and it may be applicable to other pathological models.

Phosphorescence-Assisted Microvascular O2 Measurements Reveal Alterations of Oxygen Demand in Human Metastatic Colon Cancer in the Liver of Superimmunodeficient NOG Mice

We aimed to examine metabolism of human cancer in vivo and utilized superimmunodeficient NOG mice as an experimental model of hepatic metastasis, where human colon cancer cell line HCT116 transfected with Venus, the mutant GFP was injected intrasplenically. The mice were pretreated with Pd-porphyrin to quantify local O(2) tension through intravital phosphorescence assay. In this model, a majority of metastatic foci occurred in periportal regions but not in central regions. At 1 week after the transplantation, a PO(2) drop in periportal regions was minimal without any notable decrease in microvascular blood flow. Under these conditions, there was a negative correlation between the size of metastatic foci and the lobular O(2) consumption, suggesting that the tumor O(2) consumption is smaller than that in the residual liver. At 2 weeks, portal PO(2) was significantly smaller than controls, while the central PO(2) was not comparably decreased, indicating that metastatic foci increased the O(2) consumption, while the residual liver decreased it. These results suggest metastatic tumors derived from human colon cancer exhibit notable aerobic metabolism during their developmental process, compromising respiration of the rest of the tissue regenerated during tumor development.

Facile design of plant-oil-infused fine surface asperity for transparent blood-repelling endoscope lens

Minimally invasive medical operations, especially endoscope operations, have attracted much attention and play a major role in modern medicine. Endoscope operations are superior to decrease incisions, enabling good post-operation progress. However, during its implementation, blood adheres to the lens of the endoscope, resulting in obstructed vision. This prolongs the operation time and causes the patient to gain weight. Hence, we developed a blood-repelling and transparent material for coating the surface of an endoscope lens. The coating material was produced from plant oil and a rough material for trapping the oil. Edible plant oil was particularly used to enable application to medical devices. A fine surface asperity was achieved by a one-dip treatment, which also enhanced the capillary force and durability of the oil under a water shower. The application of the developed coating material to an endoscope lens in an animal experiment enabled the effective repulsion of blood and other body fluids, the maintenance of a clear vision, and high transmittance. The developed coating material promises to contribute to the achievement of antifouling surfaces in medical devices.

Hypoxia-Inducible Factor-1 Is a Determinant of Lobular Structure and Oxygen Consumption in the Liver

Hypoxia‐inducible factor is a hypoxia‐responsive transcriptional factor that controls the expression of proteins contributing to homeostatic responses to hypoxia. Spatial heterogeneity of tissue oxygenation has been postulated as a determinant of structure and function of hepatic lobules, although its molecular mechanisms remain unknown. This study aimed to examine the role of HIF‐1 expressed in hepatocytes in regulation of hepatic microcirculation.

T-state stabilization of hemoglobin by nitric oxide to form α-nitrosyl heme causes constitutive release of ATP from human erythrocytes

Upon hypoxia, erythrocytes utilize hemoglobin (Hb) to trigger activation of glycolysis through its interaction with band 3. This process contributes to maintenance of ATP, a portion of which is released extracellularly to trigger endothelium-dependent vasorelaxation. However, whether the ATP release results either from metabolic activation of the cells secondarily or from direct regulation of the gating through Hb allostery remains unknown. This study aimed to examine if stabilization of T-state Hb could induce steady-state and hypoxia-induced alterations in glycolysis and the ATP release from erythrocytes. Treatment of deoxygenated erythrocytes with a nitric oxide (NO) donor generated alpha-NO Hb that is stabilized T-state allostery. Under these circumstances, the release of ATP was significantly elevated even under normoxia and not further enhanced upon hypoxia. These events did not coincide with activation of glycolysis of the cells, so far as judged by the fact that intracellular ATP was significantly decreased by the NO treatment. Collectively, the present study suggests that hypoxia-induced ATP release is triggered through mechanisms involving R-T transition of Hb, and the gating process might occur irrespective of hypoxia-responsive regulation of glycolysis.