Jennifer A. Fortune

Akt-Mediated Transactivation of the S1P1 Receptor in Caveolin-Enriched Microdomains Regulates Endothelial Barrier Enhancement by Oxidized Phospholipids

Endothelial cell (EC) barrier dysfunction results in increased vascular permeability, leading to increased mass transport across the vessel wall and leukocyte extravasation, the key mechanisms in pathogenesis of tissue inflammation and edema. We have previously demonstrated that OxPAPC (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine) significantly enhances vascular endothelial barrier properties in vitro and in vivo and attenuates endothelial hyperpermeability induced by inflammatory and edemagenic agents via Rac and Cdc42 GTPase dependent mechanisms. These findings suggested potential important therapeutic value of barrier-protective oxidized phospholipids. In this study, we examined involvement of signaling complexes associated with caveolin-enriched microdomains (CEMs) in barrier-protective responses of human pulmonary ECs to OxPAPC. Immunoblotting from OxPAPC-treated ECs revealed OxPAPC-mediated rapid recruitment (5 minutes) to CEMs of the sphingosine 1-phosphate receptor (S1P1), the serine/threonine kinase Akt, and the Rac1 guanine nucleotide exchange factor Tiam1 and phosphorylation of caveolin-1, indicative of signaling activation in CEMs. Abolishing CEM formation (methyl-&bgr;-cyclodextrin) blocked OxPAPC-mediated Rac1 activation, cytoskeletal reorganization, and EC barrier enhancement. Silencing (small interfering RNA) Akt expression blocked OxPAPC-mediated S1P1 activation (threonine phosphorylation), whereas silencing S1P1 receptor expression blocked OxPAPC-mediated Tiam1 recruitment to CEMs, Rac1 activation, and EC barrier enhancement. To confirm our in vitro results in an in vivo murine model of acute lung injury with pulmonary vascular hyperpermeability, we observed that selective lung silencing of caveolin-1 or S1P1 receptor expression blocked OxPAPC-mediated protection from ventilator-induced lung injury. Taken together, these results suggest Akt-dependent transactivation of S1P1 within CEMs is important for OxPAPC-mediated cortical actin rearrangement and EC barrier protection.

Atrial natriuretic peptide attenuates LPS-induced lung vascular leak: role of PAK1

Increased levels of atrial natriuretic peptide (ANP) in the models of sepsis, pulmonary edema, and acute respiratory distress syndrome (ARDS) suggest its potential role in the modulation of acute lung injury. We have recently described ANP-protective effects against thrombin-induced barrier dysfunction in pulmonary endothelial cells (EC). The current study examined involvement of the Rac effector p21-activated kinase (PAK1) in ANP-protective effects in the model of lung vascular permeability induced by bacterial wall LPS. C57BL/6J mice or ANP knockout mice (Nppa(-/-)) were treated with LPS (0.63 mg/kg intratracheal) with or without ANP (2 μg/kg iv). Lung injury was monitored by measurements of bronchoalveolar lavage protein content, cell count, Evans blue extravasation, and lung histology. Endothelial barrier properties were assessed by morphological analysis and measurements of transendothelial electrical resistance. ANP treatment stimulated Rac-dependent PAK1 phosphorylation, attenuated endothelial permeability caused by LPS, TNF-α, and IL-6, decreased LPS-induced cell and protein accumulation in bronchoalveolar lavage fluid, and suppressed Evans blue extravasation in the murine model of acute lung injury. More severe LPS-induced lung injury and vascular leak were observed in ANP knockout mice. In rescue experiments, ANP injection significantly reduced lung injury in Nppa(-/-) mice caused by LPS. Molecular inhibition of PAK1 suppressed the protective effects of ANP treatment against LPS-induced lung injury and endothelial barrier dysfunction. This study shows that the protective effects of ANP against LPS-induced vascular leak are mediated at least in part by PAK1-dependent signaling leading to EC barrier enhancement. Our data suggest a direct role for ANP in endothelial barrier regulation via modulation of small GTPase signaling.

Highly Effective Gene Transfection In Vivo by Alkylated Polyethylenimine

We mechanistically explored the effect of increased hydrophobicity of the polycation on the efficacy and specificity of gene delivery in mice. N-Alkylated linear PEIs with varying alkyl chain lengths and extent of substitution were synthesized and characterized by biophysical methods. Their in vivo transfection efficiency, specificity, and biodistribution were investigated. N-Ethylation improves the in vivo efficacy of gene expression in the mouse lung 26-fold relative to the parent polycation and more than quadruples the ratio of expression in the lung to that in all other organs. N-Propyl-PEI was the best performer in the liver and heart (581- and 3.5-fold enhancements, resp.) while N-octyl-PEI improved expression in the kidneys over the parent polymer 221-fold. As these enhancements in gene expression occur without changing the plasmid biodistribution, alkylation does not alter the cellular uptake but rather enhances transfection subsequent to cellular uptake.

Enhancement of plasmid DNA immunogenicity with linear polyethylenimine

The utility of plasmid DNA as an immunogen has been limited by its weak immunogenicity. In the present study, we evaluated the ability of a family of linear polyethylenimine (PEI) polymers, complexed to plasmid DNA, to augment DNA expression in vivo and to enhance antigen‐specific adaptive immune responses. We showed that four of five structurally different PEIs that we evaluated increased in vivo DNA expression 20‐ to 400‐fold, and enhanced DNA‐induced epitope‐specific CD8+ T‐cell responses 10‐ to 25‐fold in BALB/c and C57BL/6J mice respectively, when delivered intravenously. Functional studies of the PEI‐DNA‐induced CD8+ T‐cell responses demonstrated that formulation of DNA with PEI was associated with increased numbers of cells secreting type I cytokines. In addition, PEI‐DNA complexes improved antigen‐specific TH1‐helper cell and humoral responses. Most importantly, the PEI‐DNA complexes elicited memory cellular responses, capable of rapid expansion and accelerated clearance of a lethal dose of recombinant Listeria monocytogenes. Lastly, we identified physical properties of PEI‐DNA complexes that are associated with enhanced DNA‐elicited immunogenicity. These findings demonstrate that PEI polymers can play an important role in the development of DNA‐based vaccines in the setting of infectious disease prevention and cancer therapy.

Radio frequency radiation causes no nonthermal damage in enzymes and living cells

The ability of radio frequency radiation (RFR) to exert irreversible nonthermal (i.e., not caused by accompanying heat) effects on biologics has been widely debated due to a relative paucity of comprehensive critical details in published reports dealing with this issue. In this study, we used rigorous control over experimental conditions to determine whether continuous RFR nonthermally affects commercially important enzymes and live bacterial and human cells using three most commonly used frequencies in current RF identification technology, namely 2.45 GHz, 915 MHz, and 13.56 MHz. Diverse biological samples were exposed to RFR under deliberately harsh conditions to increase the likelihood of observing such effects should they exist. Enzymatic activities of horseradish peroxidase and β‐galactosidase in aqueous solution exhibited no statistically discernable consequences of even very intense RFR. Likewise, with putative thermal effects excluded, the viabilities of bacteria (both gram‐positive and gram‐negative) and of human cells were not detectably compromised by such an RFR exposure.

A research-inspired laboratory sequence investigating acquired drug resistance†

Here, we present a six‐session laboratory exercise designed to introduce students to standard biochemical techniques in the context of investigating a high impact research topic, acquired resistance to the cancer drug Gleevec. Students express a Gleevec‐resistant mutant of the Abelson tyrosine kinase domain, the active domain of an oncogenic protein implicated in chronic myelogenous leukemia, and investigate the kinase activity of wild type and mutant enzyme in the presence of two cancer drugs. Techniques covered include protein expression, purification, and gel analysis, kinase activity assays, and protein structure viewing. The exercises provide students with a hands‐on understanding of the impact of biochemistry on human health, and demonstrate their potential as the next generation of investigators.