Effects of cyclic strain on endothelial cell apoptosis and tubulogenesis are dependent on ROS production via NAD(P)H subunit p22phox

Objective: Vascular endothelial cells (ECs) are constantly exposed to blood flow associated forces such as cyclic strain due to blood pressure, which affects ECs survival and angiogenesis by producing ROS via NAD(P) H oxidase. NAD(P)H oxidase subunit p22phox is reported to be related to the development of atherosclerosis and increased levels of p22phox mRNA are correlated to ECs proliferation. However, the importance and signaling mechanism of p22phox on ECs survival and angiogenesis under cyclic strain are unclear. Methods: 5%-20% cyclic strain were applied by the Flexercell system to simulate in vivo environment of human ECs; the effect of p22phox on mechanical ECs survival mechanism and tubulogenesis was determined by western blot and 3-D tissue culture by knocking down p22phox expression via shRNA plasmid. Results: Knockdown of p22phox induced expression of cleaved caspase-3 and decreased cell viability ratio (CVR). 5% strain increased and 20% strain decreased CVR of shp22phox cells. There were complex biphasic effects of cyclic strain on ECs survival signaling. 5%strain continuously increased Akt phosphorylation; 20%strain increased after 10min stimulation and decreased Akt phosphorylation lately. 5% strain increased and 20% strain decreased eNOS phosphorylation. Knockdown of p22phox decreased Akt and eNOS phosphorylation with or without cyclic strain. ROS production was increasingly stimulated progressively by strain via the p22phox pathway. 5% strain increased and 20% strain decreased total NO production and vascular tubulogenesis via p22phox pathway. Conclusion: ROS production is pivotal to responses to physiological or pathological strain. Physiological strain increases but pathological strain decreases ECs survival and tubulogenesis, and these effects occur via the NAD(P) H subunit p22phox pathway. (c) 2008 Elsevier Inc. All rights reserved

Xanthine oxidase interaction with vascular endothelial growth factor in human endothelial cell angiogenesis

Objectives: Reduced capillary density occurs early in cardiovascular diseases. Oxidant stress, is implicated in endothelial apoptosis. We investigated the effects of xanthine oxidase (XO) on endothelial survival signaling: protein kinase B/Akt, its cross-talk with p38 MAPK and apoptosis pathways, and its effect on vascular tube formation in vascular endothelial growth factor (VEGF)-simulated human umbilical vein cells. Methods: We studied primary cultured human endothelial cells from the umbilical cord. Reactive oxygen species (ROS) production was detected by dibydroethidium staining, cell-signaling pathways by western blots, cell survival by western blots, and nuclear chromatin and angiogenesis response by MTT proliferation assay and three-dimensional Matrigel cultures. Results: Exogenous XO increased cellular ROS production and caused superoxide-dependent inhibition of Akt phosphorylation and enhancement of p38 MAPK phosphorylation in a time- and dose-dependent mariner. In contrast, application of the XO inhibitor oxypurinol or allopurinol inhibited VEGF-stimulated Akt phosphorylation, indicating that endogenous XO promotes VEGF-induced endothelial cell (EC) survival signaling. Exogenous XO induced activation of caspase-3 and reduced expression of the anti-apoptosis protein Bcl-2. Exogenous XO also reduced EC viability, proliferation, and vascular tube formation by p38 MAPK-dependent, phospboinositide 3-kinase (P13-K) reversible mechanisms; whereas VEGF promoted EC survival by PT3-K-dependent, p38 MAPK-independent effects. Conclusions: Exogenous XO activity is an important contributor to endothelial mechanisms for microvascular rarefaction., by modulation of cell survival signaling pathways; however, endogenons XO is necessary for maintaining EC survival

Identification and Characterization of Single-Chain Antibodies that Specifically Bind GI Noroviruses

<div><p>Norovirus infections commonly lead to outbreaks of acute gastroenteritis and spread quickly, resulting in many health and economic challenges prior to diagnosis. Rapid and reliable diagnostic tests are therefore essential to identify infections and to guide the appropriate clinical responses at the point-of-care. Existing tools, including RT-PCR and enzyme immunoassays, pose several limitations based on the significant time, equipment and expertise required to elicit results. Immunochromatographic assays available for use at the point-of-care have poor sensitivity and specificity, especially for genogroup I noroviruses, thus requiring confirmation of results with more sensitive testing methods. Therefore, there is a clear need for novel reagents to help achieve quick and reliable results. In this study, we have identified two novel single-chain antibodies (scFvs)—named NJT-R3-A2 and NJT-R3-A3—that effectively detect GI.1 and GI.7 virus-like particles (VLPs) through selection of a phage display library against the P-domain of the GI.1 major capsid protein. The limits of detection by each scFv for GI.1 and GI.7 are 0.1 and 0.2 ng, and 6.25 and 25 ng, respectively. They detect VLPs with strong specificity in multiple diagnostic formats, including ELISAs and membrane-based dot blots, and in the context of norovirus-negative stool suspensions. The scFvs also detect native virions effectively in norovirus-positive clinical stool samples. Purified scFvs bind to GI.1 and GI.7 VLPs with equilibrium constant (K_D) values of 27 nM and 49 nM, respectively. Overall, the phage-based scFv reagents identified and characterized here show utility for detecting GI.1 and GI.7 noroviruses in multiple diagnostic assay formats with strong specificity and sensitivity, indicating promise for integration into existing point-of-care tests to improve future diagnostics.</p></div

Mapping the binding of phage-displayed scFvs on the major capsid protein.

<p>Phage-displayed scFvs bind to the protruding domain (P-domain) of GI.1. Both NJT-R3-A2 and NJT-R3-A3 phages show positive binding signals against the full GI.1 VLP (black) and purified GI.1 P-domain (blue) based on signal ratios above 2 indicated by the dotted line. These signals are also statistically significant based on Fisher’s t-tests when compared to BSA where p < 0.05. Negative signals were seen for the S-domain (CT303) and negative control BSA. Each well contained the same amount of the appropriate phage at 5 × 10¹⁰ cfu/well.</p

Detection of GI.1 VLPs in dot blot format.

<p>Phage-displayed scFvs effectively detect GI.1 VLPs diluted in 10% norovirus-negative stool to amounts smaller than 0.39 ng of VLP per spot (NJT-R3-A2) and 0.78 ng (NJT-R3-A3). As a positive control, anti-NV (GI.1) polyclonal antibody showed dose-dependent positive binding signals to spots with VLPs, while negative control phage M13KE produced no binding signals for any concentration of VLPs or to stool by itself (0 ng VLP).</p

Dose response to GI.1 or GI.7 in 10% negative stool.

<p>Phage-displayed scFvs detect GI.1 and GI.7 VLPs specifically in 10% norovirus-negative stool captured by 3912 monoclonal antibodies. VLPs were diluted from a maximum of 400 ng/well to a minimum of 0.10 ng/well. The horizontal dotted line at y = 2 indicates the threshold for positive binding signals based on the definition for positive signals described earlier.</p

Binding specificity to norovirus genotypes.

<p>Binding specificity was evaluated for a panel of norovirus genotypes from genogroups GI and GII. Both NJT-R3-A2 and NJT-R3-A3 phages showed positive binding signals with antibody-captured GI.1 and GI.7, which are defined by signal ratios above 2:1 (labeled with the dotted line). These signals are also statistically significant based on t-tests where p < 0.05. No positive binding signals were observed with any of the GII genotypes tested or the BSA negative control. Each ELISA well received 5 × 10¹⁰ cfu of the appropriate phage.</p