Role of Inflammation in 20-HETE Regulation of Ischemia-Induced Angiogenesis

Objective: 20-Hydroxyeicosatetraenoic acid (20-HETE), an important bioactive lipid metabolite, has recently been identified to be a novel contributor of angiogenesis secondary to ischemia. Moreover, an inflammatory response is required for the initiation of ischemic angiogenesis, in response to ischemic tissue injury. The goal of this study is to investigate the role of inflammation in 20-HETE regulation of ischemia-induced angiogenesis. Methods: We first established a mouse hind limb ischemia model for immunocompetent Balb/C mice and immunodeficient NOD-SCID mice by femoral artery ligation. Groups of Balb/C and NOD-SCID mice were administered a 20-HETE synthesis inhibitor, DDMS, or saline as a solvent control. Laser Doppler perfusion imaging (LDPI) was used to visualize and quantify blood perfusion on days 0, 1, 3, 7, 14, and 21 post ligation, confirmed by microvessel density analysis. LC/MS/MS analysis was performed on day 3 post ligation on ischemic and non-ischemic control gracilis muscles to measure 20-HETE levels. Additionally, an antibody to lymphocyte antigen 6 complex (Ly6G/C) was administered to neutralize the infiltration of neutrophils, macrophages, and monocytes. 20-HETE levels were again measured on day 3 post ligation in these mice. Results: Quantification of the compensatory blood perfusion recovery post ischemia by LDPI showed that immunocompetent Balb/C control mice demonstrated a normal course of the compensatory angiogenic response while NOD-SCID immunodeficient mice showed a significantly decreased response. Additionally, DDMS was shown to inhibit the compensatory response in Balb/C mice, while no inhibitory effect was observed in immunodeficient NOD-SCID mice. This observation is confirmed by a marked decrease in microvessel density in SCID mice (1.5±0.2) post ischemia compared to immunocompetent Balb/C mice (2.65±0.32). As expected, ischemia markedly increased 20-HETE levels in the ischemic gracilis muscle of Balb/C mice by 6-fold (6±2 in non-ischemic vs 27±5 pg/mg in ischemic), while levels in NOD-SCID mice showed no change between the ischemic and non-ischemic control. Lastly, Balb/C mice that were treated with Ly6G/C neutralizing antibody exhibited significantly decreased 20-HETE levels in their ischemic gracilis muscle compared to the non-ischemic control. Conclusion: Inflammation may be an essential contributor in 20-HETE regulation of the ischemia-induced angiogenic response

Combination of Vatalanib and a 20-HETE Synthesis Inhibitor Results in Decreased Tumor Growth in an Animal Model of Human Glioma

BACKGROUND: Due to the hypervascular nature of glioblastoma (GBM), antiangiogenic treatments, such as vatalanib, have been added as an adjuvant to control angiogenesis and tumor growth. However, evidence of progressive tumor growth and resistance to antiangiogenic treatment has been observed. To counter the unwanted effect of vatalanib on GBM growth, we have added a new agent known as N-hydroxy-N\u27-(4-butyl-2 methylphenyl)formamidine (HET0016), which is a selective inhibitor of 20-hydroxyeicosatetraenoic acid (20-HETE) synthesis. The aims of the studies were to determine 1) whether the addition of HET0016 can attenuate the unwanted effect of vatalanib on tumor growth and 2) whether the treatment schedule would have a crucial impact on controlling GBM. METHODS: U251 human glioma cells (4×10(5)) were implanted orthotopically. Two different treatment schedules were investigated. Treatment starting on day 8 (8-21 days treatment) of the tumor implantation was to mimic treatment following detection of tumor, where tumor would have hypoxic microenvironment and well-developed neovascularization. Drug treatment starting on the same day of tumor implantation (0-21 days treatment) was to mimic cases following radiation therapy or surgery. There were four different treatment groups: vehicle, vatalanib (oral treatment 50 mg/kg/d), HET0016 (intraperitoneal treatment 10 mg/kg/d), and combined (vatalanib and HET0016). Following scheduled treatments, all animals underwent magnetic resonance imaging on day 22, followed by euthanasia. Brain specimens were equally divided for immunohistochemistry and protein array analysis. RESULTS: Our results demonstrated a trend that HET0016, alone or in combination with vatalanib, is capable of controlling the tumor growth compared with that of vatalanib alone, indicating attenuation of the unwanted effect of vatalanib. When both vatalanib and HET0016 were administered together on the day of the tumor implantation (0-21 days treatment), tumor volume, tumor blood volume, permeability, extravascular and extracellular space volume, tumor cell proliferation, and cell migration were decreased compared with that of the vehicle-treated group. CONCLUSION: HET0016 is capable of controlling tumor growth and migration, but these effects are dependent on the timing of drug administration. The addition of HET0016 to vatalanib may attenuate the unwanted effect of vatalanib

Intravenous Formulation of HET0016 Decreased Human Glioblastoma Growth and Iimplicated Survival Benefit in Rat Xenograft Models

Glioblastoma (GBM) is a hypervascular primary brain tumor with poor prognosis. HET0016 is a selective CYP450 inhibitor, which has been shown to inhibit angiogenesis and tumor growth. Therefore, to explore novel treatments, we have generated an improved intravenous (IV) formulation of HET0016 with HPssCD and tested in animal models of human and syngeneic GBM. Administration of a single IV dose resulted in 7-fold higher levels of HET0016 in plasma and 3.6-fold higher levels in tumor at 60 min than that in IP route. IV treatment with HPssCD-HET0016 decreased tumor growth, and altered vascular kinetics in early and late treatment groups (p \u3c 0.05). Similar growth inhibition was observed in syngeneic GL261 GBM (p \u3c 0.05). Survival studies using patient derived xenografts of GBM811, showed prolonged survival to 26 weeks in animals treated with focal radiation, in combination with HET0016 and TMZ (p \u3c 0.05). We observed reduced expression of markers of cell proliferation (Ki-67), decreased neovascularization (laminin and alphaSMA), in addition to inflammation and angiogenesis markers in the treatment group (p \u3c 0.05). Our results indicate that HPssCD-HET0016 is effective in inhibiting tumor growth through decreasing proliferation, and neovascularization. Furthermore, HPssCD-HET0016 significantly prolonged survival in PDX GBM811 model

Human Cord Blood-Derived AC133+ Progenitor Cells Preserve Endothelial Progenitor Characteristics after Long Term In Vitro Expansion

Stem cells/progenitors are central to the development of cell therapy approaches for vascular ischemic diseases. The crucial step in rescuing tissues from ischemia is improvement of vascularization that can be achieved by promoting neovascularization. Endothelial progenitor cells (EPCs) are the best candidates for developing such an approach due to their ability to self-renew, circulate and differentiate into mature endothelial cells (ECs). Studies showed that intravenously administered progenitors isolated from bone marrow, peripheral or cord blood home to ischemic sites. However, the successful clinical application of such transplantation therapy is limited by low quantities of EPCs that can be generated from patients. Hence, the ability to amplify the numbers of autologous EPCs by long term in vitro expansion while preserving their angiogenic potential is critically important for developing EPC based therapies. Therefore, the objective of this study was to evaluate the capacity of cord blood (CB)-derived AC133+ cells to differentiate, in vitro, towards functional, mature endothelial cells (ECs) after long term in vitro expansion.We systematically characterized the properties of CB AC133+ cells over the 30 days of in vitro expansion. During 30 days of culturing, CB AC133+ cells exhibited significant growth potential that was manifested as 148-fold increase in cell numbers. Flow cytometry and immunocytochemistry demonstrated that CB AC133+ cells' expression of endothelial progenitor markers was not affected by long term in vitro culturing. After culturing under EC differentiation conditions, cells exhibited high expression of mature ECs markers, such as CD31, VEGFR-2 and von Willebrand factor, as well as the morphological changes indicative of differentiation towards mature ECs. In addition, throughout the 30 day culture cells preserved their functional capacity that was demonstrated by high uptake of DiI fluorescently conjugated-acetylated-low density lipoprotein (DiI-Ac-LDL), in vitro and in vivo migration towards chemotactic stimuli and in vitro tube formation.These studies demonstrate that primary CB AC133+ culture contained mainly EPCs and that long term in vitro conditions facilitated the maintenance of these cells in the state of commitment towards endothelial lineage

Predominant Expression of Hybrid N-Glycans Has Distinct Cellular Roles Relative to Complex and Oligomannose N-Glycans

Glycosylation modulates growth, maintenance, and stress signaling processes. Consequently, altered N-glycosylation is associated with reduced fitness and disease. Therefore, expanding our understanding of N-glycans in altering biological processes is of utmost interest. Herein, clustered regularly interspaced short palindromic repeats/caspase9 (CRISPR/Cas9) technology was employed to engineer a glycosylation mutant Chinese Hamster Ovary (CHO) cell line, K16, which expresses predominantly hybrid type N-glycans. This newly engineered cell line enabled us to compare N-glycan effects on cellular properties of hybrid type N-glycans, to the well-established Pro´5 and Lec1 cell lines, which express complex and oligomannose types of N-glycans, respectively. Lectin binding studies revealed the predominant N-glycan expressed in K16 is hybrid type. Cell dissociation and migration assays demonstrated the greatest strength of cell–cell adhesion and fastest migratory rates for oligomannose N-glycans, and these properties decreased as oligomannose type were converted to hybrid type, and further decreased upon conversion to complex type. Next, we examined the roles of three general types of N-glycans on ectopic expression of E-cadherin, a cell–cell adhesion protein. Microscopy revealed more functional E-cadherin at the cell–cell border when N-glycans were oligomannose and these levels decreased as the oligomannose N-glycans were processed to hybrid and then to complex. Thus, we provide evidence that all three general types of N-glycans impact plasma membrane architecture and cellular properti

The Effect of the Crow Hop on Elbow Stress During an Interval Throwing Program

BACKGROUND: Postoperative rehabilitation protocols after ulnar collateral ligament (UCL) reconstruction typically involve a structured interval throwing program. In an effort to minimize torque placed on the UCL, athletes are often instructed to throw with a crow hop, even at short throwing distances. However, the effect of the crow hop on medial elbow stress is unknown. PURPOSE/HYPOTHESIS: The purpose was to determine whether elbow stress differs with and without a crow hop across the throwing distances of a typical interval throwing program. We hypothesized that crow hop throws would generate lower torque on the elbow than standing throws at each distance of the interval throwing program. STUDY DESIGN: Controlled laboratory study. METHODS: Healthy high school and collegiate pitchers and position players were recruited from the surrounding area. Each player was outfitted with a wearable athletic sleeve and device that recorded elbow torque (Newton-meters), arm slot (degrees), arm speed (revolutions per minute), and shoulder rotation (degrees). Ball velocity (miles per hour) was measured using a radar gun. Players were instructed to perform 3 crow hop throws and 3 standing throws at distances of 30, 45, 60, 90, 120, 150, and 180 feet. A repeated measures analysis of variance was used to compare ball velocity, elbow torque, arm slot, arm speed, and shoulder rotation between crow hop and standing throws at each throwing distance. RESULTS: Twenty athletes participated in this study (average age, 17.8 years; range, 15-25 years). The average medial elbow torque increased at each distance for both crow hop and standing throws at distances of 30, 45, 60, and 90 feet (P \u3c .05), after which there were no significant increases in elbow torque (P \u3e .05). The average torque was higher for crow hop throws than standing throws at distances of 30 feet (13.9 N·m vs 12.0 N·m; P = .002), 45 feet (21.8 N·m vs 19.3 N·m; P = .005), and 60 feet (28.0 N·m vs 24.5 N·m; P = .02). CONCLUSION: Crow hop throws generated greater medial elbow torque than standing throws at distances up to 60 feet; however, there were no differences in elbow torque at distances greater than 60 feet between the 2 throw types. For both crow hop and standing throws, elbow stress increased at each distance interval up to 90 feet before plateauing at distances greater than 90 feet. The crow hop throwing technique does not reduce medial elbow stress during a simulated interval throwing program, and it may actually increase torque at shorter throwing distances. CLINICAL RELEVANCE: The results of our study indicate that it would be prudent for players to initially perform standing throws at shorter distances and only later be allowed to employ a natural crow hop at greater distances to minimize torque placed on the medial elbow during UCL rehabilitation protocols

Electronic structure investigation of CoO by means of soft X-ray scattering

The electronic structure of CoO is studied by resonant inelastic soft X-ray scattering spectroscopy using photon energies across the Co 2p absorption edges. The different spectral contributions from the energy-loss structures are identified as Raman scattering due to d-d and charge-transfer excitations. For excitation energies close to the L3 resonance, the spectral features are dominated by quartet-quartet and quartet-doublet transitions of the 3d7 configuration. At excitation energies corresponding to the satellites in the Co 2p X-ray absorption spectrum of CoO, the emission features are instead dominated by charge-transfer transitions to the 3d8L-1 final state. The spectra are interpreted and discussed with the support of simulations within the single impurity Anderson model with full multiplet effects which are found to yield consistent spectral functions to the experimental data.Comment: 8 pages, 2 figures, 2 tables, http://link.aps.org/doi/10.1103/PhysRevB.65.20510

Low-cost, Transportable Hydrogen Fueling Station for Early FCEV Adoption

Thousands of public hydrogen fueling stations are needed to support the early Fuel Cell Electric Vehicle (FCEV) market in the U.S.; there are currently 12. The California state government has been the largest investor of the hydrogen fueling infrastructure funding 9 permanent stations currently open to the public with 48 more in development costing anywhere from $1.8M-$5.5M each. To attract private investors and decrease dependence on government funding, a low-cost, mobile hydrogen dispensing system must be developed. This paper describes a transportable hydrogen fueling station that has been designed for $423,000 using off-the-shelf components, less than 23$9.62/kg. This paper presents the mechanical design and operation of the fueling station. A complete report including an economic analysis and safety features is available at: http://hydrogencontest.org/pdf/2014/WSU_2014_HEF_CONTEST.pdf

Evidence for the immobile bipolaron formation in the paramagnetic state of the magnetoresistive manganites

Recent research suggests that the charge carriers in the paramagnetic state of the magnetoresistive manganites are small polarons. Here we report studies of the oxygen-isotope effects on the intrinsic resistivity and thermoelectric power in several ferromagnetic manganites. The precise measurements of these isotope effects allow us to make a quantitative data analysis. Our results do not support a simple small-polaron model, but rather provide compelling evidence for the presence of small immobile bipolarons, i.e., pairs of small polarons. Since the bipolarons in the manganites are immobile, the present result alone appears not to give a positive support to the bipolaronic superconductivity theory for the copper-based perovskites.Comment: 6 pages, 5 figures, monor correction

Role of mitochondrial raft-like microdomains in the regulation of cell apoptosis

Lipid rafts are envisaged as lateral assemblies of specific lipids and proteins that dissociate and associate rapidly and form functional clusters in cell membranes. These structural platforms are not confined to the plasma membrane; indeed lipid microdomains are similarly formed at subcellular organelles, which include endoplasmic reticulum, Golgi and mitochondria, named raft-like microdomains. In addition, some components of raft-like microdomains are present within ER-mitochondria associated membranes. This review is focused on the role of mitochondrial raft-like microdomains in the regulation of cell apoptosis, since these microdomains may represent preferential sites where key reactions take place, regulating mitochondria hyperpolarization, fission-associated changes, megapore formation and release of apoptogenic factors. These structural platforms appear to modulate cytoplasmic pathways switching cell fate towards cell survival or death. Main insights on this issue derive from some pathological conditions in which alterations of microdomains structure or function can lead to severe alterations of cell activity and life span. In the light of the role played by raft-like microdomains to integrate apoptotic signals and in regulating mitochondrial dynamics, it is conceivable that these membrane structures may play a role in the mitochondrial alterations observed in some of the most common human neurodegenerative diseases, such as Amyotrophic lateral sclerosis, Huntington's chorea and prion-related diseases. These findings introduce an additional task for identifying new molecular target(s) of pharmacological agents in these pathologies