Prostaglandin E 2 (PGE 2 ) and prostacyclin are lipid mediators produced by cyclooxygenase and implicated in the regulation of vascular function, wound repair, inflammatory processes, and acute lung injury. Although protective effects of these prostaglandins (PGs) are associated with stimulation of intracellular cAMP production, the crosstalk between cAMP-activated signal pathways in the regulation of endothelial cell (EC) permeability is not well understood. We studied involvement of cAMP-dependent kinase (PKA), cAMP-Epac-Rap1 pathway, and small GTPase Rac in the PGsinduced EC barrier protective effects and cytoskeletal remodeling. PGE 2 and PGI 2 synthetic analog beraprost increased transendothelial electrical resistance and decreased dextran permeability, enhanced peripheral F-actin rim and increased intercellular adherens junction areas reflecting EC barrier-protective response. Furthermore, beraprost dramatically attenuated thrombin-induced Rho activation, MLC phosphorylation and EC barrier dysfunction. In vivo, beraprost attenuated lung barrier dysfunction induced by high tidal volume mechanical ventilation. Both PGs caused cAMPmediated activation of PKA-, Epac/Rap1-and Tiam1/Vav2-dependent pathways of Rac1 activation and EC barrier regulation. Knockdown of Epac, Rap1, Rac-specific exchange factors Tiam1 and Vav2 using siRNA approach, or inhibition of PKA activity decreased Rac1 activation and PGinduced EC barrier enhancement. Thus, our results show that barrier-protective effects of PGE 2 and prostacyclin on pulmonary EC are mediated by PKA and Epac/Rap pathways, which converge on Rac activation and lead to enhancement of peripheral actin cytoskeleton and adherens junctions. These mechanisms may mediate protective effects of PGs against agonist-induced lung vascular barrier dysfunction in vitro and against mechanical stress-induced lung injury in vivo.

Lack of knowledge about how regulatory regions evolve in relation to their structure–function may limit the utility of comparative sequence analysis in deciphering cis-regulatory sequences. To address this we applied reverse genetics to carry out a functional genetic complementation analysis of a eukaryotic cis-regulatory module—the even-skipped stripe 2 enhancer—from four Drosophila species. The evolution of this enhancer is non-clock-like, with important functional differences between closely related species and functional convergence between distantly related species. Functional divergence is attributable to differences in activation levels rather than spatiotemporal control of gene expression. Our findings have implications for understanding enhancer structure–function, mechanisms of speciation and computational identification of regulatory modules.

Segmentation in Drosophila embryogenesis occurs through a hierarchical cascade of regulatory gene expression driven by the establishment of a diffusion-mediated morphogen gradient. Here, we investigate the response of this pattern formation process to genetic variation and evolution in egg size. Specifically, we ask whether spatial localization of gap genes Kruppel (Kr) and giant (gt) and the pair-rule gene even-skipped (eve) during cellularization is robust to genetic variation in embryo length in three Drosophila melanogaster isolines and two closely related species. We identified two wild-derived strains of D. melanogaster whose eggs differ by Ϸ25% in length when reared under identical conditions. These two lines, a D. melanogaster laboratory stock (w1118), and offspring from crosses between the lines all exhibit precise scaling in the placement of gap and pair-rule gene expression along the anterior-posterior axis in relation to embryo length. Genetic analysis indicates that this scaling is maternally controlled. Maternal regulation of scaling must be required for consistent localization of segmentation gene expression because embryo size, a genetically variable and adaptive trait, is maternally inherited. We also investigated spatial scaling between these D. melanogaster lines and single lines of Drosophila sechellia and Drosophila simulans, the latter two differing by Ϸ25% in egg length. In contrast to the robust scaling we observed within species, localization of gene expression relative to embryo length differs significantly between the three species. Thus, the developmental mechanism that assures robust scaling within a species does not prevent rapid evolution between species.buffering ͉ development ͉ embryo size ͉ genetic variation ͉ scaling

Acute lung injury, sepsis, lung inflammation, and ventilator-induced lung injury are life-threatening conditions associated with lung vascular barrier dysfunction, which may lead to pulmonary edema. Increased levels of atrial natriuretic peptide (ANP) in lung circulation reported in these pathologies suggest its potential role in the modulation of lung injury. Besides well recognized physiological effects on vascular tone, plasma volume, and renal function, ANP may exhibit protective effects in models of lung vascular endothelial cell (EC) barrier dysfunction. However, the molecular mechanisms of ANP protective effects are not well understood. The recently described cAMP-dependent guanine nucleotide exchange factor (GEF) Epac activates small GTPase Rap1, which results in activation of small GTPase Rac-specific GEFs Tiam1 and Vav2 and Rac-mediated EC barrier protective responses. Our results show that ANP stimulated protein kinase A and the Epac/Rap1/Tiam/Vav/Rac cascade dramatically attenuated thrombin-induced pulmonary EC permeability and the disruption of EC monolayer integrity. Using pharmacological and molecular activation and inhibition of cAMP-and cGMP-dependent protein kinases (PKA and PKG), Epac, Rap1, Tiam1, Vav2, and Rac we linked ANP-mediated protective effects to the activation of Epac/Rap and PKA signaling cascades, which dramatically inhibited the Rho pathway of thrombin-induced EC hyper-permeability. These results suggest a novel mechanism of ANP protective effects against agonist-induced pulmonary EC barrier dysfunction via inhibition of Rho signaling by Epac/Rap1-Rac and PKA signaling cascades.

Reorganization of the endothelial cell (EC) cytoskeleton and cell adhesive complexes provides a structural basis for increased vascular permeability implicated in the pathogenesis of many diseases, including asthma, sepsis, and acute respiratory distress syndrome (ARDS). We have recently described the barrier-protective effects of hepatocyte growth factor (HGF) on the human pulmonary EC. In the present study, we explored the involvement of Rac-GTPase and Rac-specific nucleotide exchange factor Tiam1 in the mechanisms of EC barrier protection by HGF. HGF protected EC monolayers from thrombin-induced hyperpermeability, disruption of intercellular junctions, and formation of stress fibers and paracellular gaps by inhibiting thrombin-induced activation of Rho GTPase, Rho association with nucleotide exchange factor p115-RhoGEF, and myosin light chain phosphorylation, which was opposed by stimulation of Rac-dependent signaling. The pharmacological Rac inhibitor or silencing RNA (siRNA) based depletion of either Rac or Tiam1 significantly attenuated HGF-induced peripheral translocation of Rac effector cortactin, cortical actin ring formation, and EC barrier enhancement. Moreover, Tiam1 knockdown using the siRNA approach, attenuated the protective effect of HGF against thrombin-induced activation of Rho signaling, monolayer disruption, and EC hyperpermeability. This study demonstrates the Tiam1/Rac-dependent mechanism of HGF-induced EC barrier protection and provides novel mechanistic insights into regulation of EC permeability via dynamic interactions between Rho- and Tiam1/Rac-mediated pathways.