Loss of fluid shear stress (ischemia) to the lung endothelium causes endothelial plasma membrane depolarization via ATP-sensitive K(+) (K(ATP)) channel closure, initiating a signaling cascade that leads to NADPH oxidase (NOX2) activation and ROS production. Since wortmannin treatment significantly reduces ROS production with ischemia, we investigated the role of phosphoinositide 3-kinase (PI3K) in shear-associated signaling. Pulmonary microvascular endothelial cells in perfused lungs subjected to abrupt stop of flow showed membrane depolarization and ROS generation. Stop of flow in flow-adapted mouse pulmonary microvascular endothelial cells in vitro resulted in the activation of PI3K and Akt as well as ROS generation. ROS generation in the lungs in situ was almost abolished by the PI3K inhibitor wortmannin and the PKC inhibitor H7. The combination of the two (wortmannin and H7) did not have a greater effect. Activation of NOX2 was greatly diminished by wortmannin, knockout of Akt1, or dominant negative PI3K, whereas membrane depolarization was unaffected. Ischemia-induced Akt activation (phosphorylation) was not observed with K(ATP) channel-null cells, which showed minimal changes in membrane potential with ischemia. Activation of Akt was similar to wild-type cells in NOX2-null cells, which do not generate ROS with ischemia. Cromakalim, a K(ATP) channel agonist, prevented both membrane depolarization and Akt phosphorylation with ischemia. Thus, Akt1 phosphorylation follows cell membrane depolarization and precedes the activation of NOX2. These results indicate that PI3K/Akt and PKC serve as mediators between endothelial cell membrane depolarization and NOX2 assembly.

Aims: We reported earlier that ischemia results in the generation of reactive oxygen species (ROS) via the closure of a K ATP channel which causes membrane depolarization and NADPH oxidase 2 (NOX2) activation. This study was undertaken to understand the role of ischemia-mediated ROS in signaling. Results: Angiogenic potential of pulmonary microvascular endothelial cells (PMVEC) was studied in vitro and in the hind limb in vivo. Flow adapted PMVEC injected into a Matrigel matrix showed significantly higher tube formation than cells grown under static conditions or cells from mice with knockout of K ATP channels or the NOX2. Blocking of hypoxia inducible factor-1 alpha (HIF-1a) accumulation completely abrogated the tube formation in wild-type (WT) PMVEC. With ischemia in vivo (femoral artery ligation), revascularization was high in WT mice and was significantly decreased in mice with knockout of K ATP channel and in mice orally fed with a K ATP channel agonist. In transgenic mice with endothelial-specific NOX2 expression, the revascularization observed was intermediate between that of WT and knockout of K ATP channel or NOX2. Increased HIF-1a activation and vascular endothelial growth factor (VEGF) expression was observed in ischemic tissue of WT mice but not in K ATP channel and NOX2 null mice. Revascularization could be partially rescued in K ATP channel null mice by delivering VEGF into the hind limb. Innovation: This is the first report of a mechanosensitive ion channel (K ATP channel) initiating endothelial signaling that drives revascularization. Conclusion: The K ATP channel responds to the stop of flow and activates signals for revascularization to restore the impeded blood flow. Antioxid. Redox Signal. 20,[872][873][874][875][876][877][878][879][880][881][882][883][884][885][886]

The lung endothelium is exposed to mechanical stimuli through shear stress arising from blood flow and responds to altered shear by activation of NADPH (NOX2) to generate reactive oxygen species (ROS). This review describes the pathway for NOX2 activation and the downstream ROS-mediated signaling events on the basis of studies of isolated lungs and flow-adapted endothelial cells in vitro that are subjected to acute flow cessation (ischemia). Altered mechanical stress is detected by a cell-associated complex involving caveolae and other membrane proteins that results in endothelial cell membrane depolarization and then the activation of specific kinases that lead to the assembly of NOX2 components. ROS generated by this enzyme amplify the mechanosignal within the endothelial cell to regulate activation and/or synthesis of proteins that participate in cell growth, proliferation, differentiation, apoptosis, and vascular remodeling. These responses indicate an important role for NOX2-derived ROS associated with mechanotransduction in promoting vascular homeostasis.

Lipids represent a diverse array of molecules essential to the cell's structure, defense, energy, and communication. Lipid metabolism can often become dysregulated during tumor development. During cancer therapy, targeted inhibition of cell proliferation can likewise cause widespread and drastic changes in lipid composition. Molecular imaging techniques have been developed to monitor altered lipid profiles as a biomarker for cancer diagnosis and treatment response. For decades, MRS has been the dominant non-invasive technique for studying lipid metabolite levels. Recent insights into the oncogenic transformations driving changes in lipid metabolism haverevealed new mechanisms and signaling molecules that can be exploited using optical imaging, mass spectrometry imaging, and positron emission tomography. These novel imaging modalities have provided researchers with a diverse toolbox to examine changes in lipids in response to a wide array of anticancer strategies including chemotherapy, radiation therapy, signal transduction inhibitors, gene therapy, immunotherapy, or a combination of these strategies. The understanding of lipid metabolism in response to cancer therapy continues to evolve as each therapeutic method emerges, and this review seeks to summarize the current field and areas of unmet needs.

In this article we review methods for the study of perfused cells, tissues, and organs using magnetic resonance spectroscopy (MRS). Biologically relevant nuclei and the different but often complementary information that they make available are presented. Pulse sequence implementation is discussed in the context of tailoring the desired metabolite information and suppressing unwanted resonances. Metabolites observable with MRS are generally limited to those present in relatively high (millimolar) concentrations. The importance of these metabolites and how flux through metabolic pathways can be observed using isotopic enhancement with 13 C is reviewed. Cell preparation and immobilization methods employing agarose threads, matrigel and alginate beads; and the use of porous and nonporous microcarriers, hollow fibers and bioreactors for perfused cell culture are discussed. Tissue preparation techniques for the study of perfused organs include the Langendorff heart, perfused liver, and lung preparations. Maintaining careful control of the perfusate composition, pH, oxygenation, and temperature all critically affect cell and organ viability as well as the measured levels of cell metabolites. The requisite apparatus for achieving this is reviewed. Perfusion model systems allow the acquisition of real-time kinetic metabolic information by MRS, providing unique insights into the effects of drugs and inhibitors on metabolite levels. They thus provide a valuable intermediate for translational research from cells to animals to humans and back again.

No abstract

No abstract

We reported earlier that ischemia or loss of shear in the lung endothelium causes endothelial membrane depolarization (via KATP channel closure) that leads to NADPH oxidase activation and reactive oxygen species (ROS) production. ROS production was abolished in the presence of PI3Kinase/Akt inhibitor, wortmanin. We thus hypothesized that PI3K/Akt activation is the trigger for ROS production with pulmonary ischemia. We used pulmonary microvascular endothelial cells, either wild type (WT), KATP null (KIR6.2−/−), NADPH oxidase null (gp91phox−/−) or WT infected with dominant negative (DN) PI3K, and isolated WT and Akt‐1 null lungs to study signal transduction with ischemia. In WT lungs, the PI3K inhibitor wortmannin had no effect on ischemia mediated depolarization. Cells with DN PI3K and lungs with Akt1−/− depolarized with ischemia but failed to show NADPH oxidase assembly and ROS generation. Depolarization in WT and Akt−/− lungs was blocked upon addition of cromakalim (KATP channel agonist). In WT cells, Akt phosphorylation that was observed with ischemia and was abolished in KIR6.2−/− but not in gp91phox−/− cells. These results indicate that KATP induced depolarization leads to PI3K/Akt activation that triggers NADPH oxidase assembly and eventual ROS generation. Thus, ROS which has hitherto been shown to activate PI3K/Akt in other systems lies downstream of PI3K/Akt in our model of pulmonary ischemia.