Recent work shows that cooling protection after mouse cardiac arrest and cardiomyocyte ischemia is mediated by Akt activation. The PI3K p85 subunit can either augment or inhibit Akt activation depending on its binding to p110 or PTEN respectively. To further clarify the role of PI3K p85 in cardioprotection, we studied novel TAT-p85 fusion proteins that selectively inhibit PI3K p85 binding. We hypothesized that TAT fused p85 lacking the PTEN binding site (TAT-?PTEN p85) would enhance Akt phosphorylation to afford cardioprotection. Conversely, TAT fused p85 lacking the p110 binding site (TAT-?p110p85) would decrease Akt phosphorylation and abrogate cardioprotection. Microscopy and Western blot analysis demonstrated that TAT fusion protein was transduced into cardiomyocytes within 5 min and remained more than 2 h. Inhibition of PI3K/Akt by TAT-?p110 p85 significantly increased cell death from 44.6±2.7% to 92.5±3.4% after simulated ischemia and reperfusion. By contrast, PTEN inhibition using TAT-?PTEN p85 decreased cell death to 11.9±5.3%, a similar level of cardioprotection seen with past cooling studies. Additional studies with the small molecule PTEN inhibitor VO-OHpic confirmed that PTEN inhibition was highly protective against cell death induced by ischemia and reperfusion. We conclude that blockade of p85-PTEN interaction and PTEN inhibition may be promising strategies for rescuing the heart from ischemia and reperfusion injury.
Ventilation at high tidal volume may cause lung inflammation and barrier dysfunction that culminates in ventilator-induced lung injury (VILI). However, the mechanisms by which mechanical stimulation triggers the inflammatory response have not been fully elucidated. This study tested the hypothesis that onset of VILI is triggered by activation of secretory group V phospholipase A(2) (gVPLA2) in pulmonary vascular endothelium exposed to excessive mechanical stretch. High-magnitude cyclic stretch (18% CS) increased expression and surface exposure of gVPLA2 in human pulmonary endothelial cells (EC). CS-induced gVPLA2 activation was required for activation of ICAM-1 expression and polymorphonuclear neutrophil (PMN) adhesion to CS-preconditioned EC. By contrast, physiological CS (5% CS) had no effect on gVPLA2 activation or EC-PMN adhesion. CS-induced ICAM-1 expression and EC-PMN adhesion were attenuated by the gVPLA2-blocking antibody (MCL-3G1), general inhibitor of soluble PLA2, LY311727, or siRNA-induced EC gVPLA2 knockdown. In vivo, ventilator-induced lung leukocyte recruitment, cell and protein accumulation in the alveolar space, and total lung myeloperoxidase activity were strongly suppressed in gVPLA2 mouse knockout model or upon administration of MCL-3G1. These results demonstrate a novel role for gVPLA2 as the downstream effector of pathological mechanical stretch leading to an inflammatory response associated with VILI.
Cytosolic gIVaPLA2 is a critical enzyme in the generation of arachidonate metabolites and in induction of beta2-integrin adhesion in granulocytes. We hypothesized that gIVaPLA2 activation also is an essential downstream step for post adhesive migration of PMN in vitro.
The Forkhead Box m1 (Foxm1) protein is induced in a majority of human non-small cell lung cancers and its expression is associated with poor prognosis. However, specific requirements for the Foxm1 in each cell type of the cancer lesion remain unknown. The present study provides the first genetic evidence that the Foxm1 expression in respiratory epithelial cells is essential for lung tumorigenesis. Using transgenic mice, we demonstrated that conditional deletion of Foxm1 from lung epithelial cells (epFoxm1(-/-) mice) prior to tumor initiation caused a striking reduction in the number and size of lung tumors, induced by either urethane or 3-methylcholanthrene (MCA)/butylated hydroxytoluene (BHT). Decreased lung tumorigenesis in epFoxm1(-/-) mice was associated with diminished proliferation of tumor cells and reduced expression of Topoisomerase-2alpha (TOPO-2alpha), a critical regulator of tumor cell proliferation. Depletion of Foxm1 mRNA in cultured lung adenocarcinoma cells significantly decreased TOPO-2alpha mRNA and protein levels. Moreover, Foxm1 directly bound to and induced transcription of the mouse TOPO-2alpha promoter region, indicating that TOPO-2alpha is a direct target of Foxm1 in lung tumor cells. Finally, we demonstrated that a conditional deletion of Foxm1 in pre-existing lung tumors dramatically reduced tumor growth in the lung. Expression of Foxm1 in respiratory epithelial cells is critical for lung cancer formation and TOPO-2alpha expression in vivo, suggesting that Foxm1 is a promising target for anti-tumor therapy.
We investigated the regulatory role of 14-kDa secretory group V phospholipase A(2) (gVPLA(2)) in the development of acute lung injury (ALI) and neutrophilic inflammation (NI) caused by intratracheal administration of LPS. Experiments were conducted in gVPLA(2) knockout (pla2g5(-/-)) mice, which lack the gene, and gVPLA(2) wild-type littermate control (pla2g5(+/+)) mice. Indices of pulmonary injury were evaluated 24 h after intratracheal administration of LPS. Expression of gVPLA(2) in microsections of airways and mRNA content in lung homogenates were increased substantially in pla2g5(+/+) mice after LPS-administered compared with saline-treated pla2g5(+/+) mice. By contrast, expression of gVPLA(2) was neither localized in LPS- nor saline-treated pla2g5(-/-) mice. LPS also caused 1) reduced transthoracic static compliance, 2) lung edema, 3) neutrophilic infiltration, and 4) increased neutrophil myeloperoxidase activity in pla2g5(+/+) mice. These events were attenuated in pla2g5(-/-) mice exposed to LPS or in pla2g5(+/+) mice receiving MCL-3G1, a neutralizing MAb directed against gVPLA(2), before LPS administration. Our data demonstrate that gVPLA(2) is an inducible protein in pla2g5(+/+) mice but not in pla2g5(-/-) mice within 24 h after LPS treatment. Specific inhibition of gVPLA(2) with MCL-3G1 or gene-targeted mice lacking gVPLA(2) blocks ALI and attenuates NI caused by LPS.
Acute lung injury (ALI) is characterized by inflammatory disruption of the alveolar-vascular barrier, resulting in severe respiratory compromise. Inhibition of the intercellular messenger protein, Group V phospholipase A(2) (gVPLA(2)), blocks vascular permeability caused by LPS both in vivo and in vitro. In this investigation we studied the mechanism by which recombinant gVPLA(2) increases permeability of cultured human pulmonary endothelial cells (EC). Exogenous gVPLA(2) (500 nM), a highly hydrolytic enzyme, caused a significant increase in EC permeability that began within minutes and persisted for >10 hours. However, the major hydrolysis products of gVPLA(2) (Lyso-PC, Lyso-PG, LPA, arachidonic acid) did not cause EC structural rearrangement or loss of barrier function at concentrations <10 ?M. Higher concentrations (? 30 ?M) of these membrane hydrolysis products caused some increased permeability but were associated with EC toxicity (measured by propidium iodide incorporation) that did not occur with barrier disruption by gVPLA(2) (500 nM). Pharmacologic inhibition of multiple intracellular signaling pathways induced by gVPLA(2) activity (ERK, p38, PI3K, cytosolic gIVPLA(2)) also did not prevent EC barrier disruption by gVPLA(2). Finally, pretreatment with heparinase to prevent internalization of gVPLA(2) did not inhibit EC barrier disruption by gVPLA(2). Our data thus indicate that gVPLA(2) increases pulmonary EC permeability directly through action as a membrane hydrolytic agent. Disruption of EC barrier function does not depend upon membrane hydrolysis products, gVPLA(2) internalization, or upregulation of downstream intracellular signaling.
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