Introduction: Recent work shows that PTEN inhibition protects against cardiomyocyte contractile dysfunction and death following ischemia/reperfusion. To further determine whether PTEN inhibition improves metabolic recovery and survival in a mouse model of cardiac arrest, we have designed a TAT fused cell-permeable peptide (TAT-PTEN9c) based on the c-terminal PDZ binding motif of PTEN for rapid tissue delivery. We hypothesized that TAT-PTEN9c interferes with endogenous PTEN binding to cell membrane adaptors resulting in increased Akt activation, causing Akt-enhanced glucose utilization (with decreased diversion of glucose via the alternate polyol pathway to sorbitol) and improved survival. Methods: Mouse cardiomyocytes were isolated from 1-3 day old mouse pups. Western blot was used to identify concentrations of TAT-PTEN9c that enhanced Akt phosphorylation in mouse cardiomyocytes exposed to oxidant stress. C57BL6 mice were then subjected to an established KCL-induced 8 min cardiac arrest protocol. 30 mice after CPR were randomly assigned to receive saline or TAT-PTEN9c. MAP, ETCO2, and ECG were recorded until 4 h after successful cardiopulmonary resuscitation (CPR). TAT-PTEN9c (7.8 mg/kg) was given intravenously (IV) after CPR (n=10). As a measure of impaired glucose utilization, sorbitol content in heart and brain was determined by a fluorescence assay of NADH formation using sorbitol dehydrogenase and NAD+. Results: TAT-PTEN9c peptide enhanced Akt activation in neonatal mouse cardiomyoctes in a concentration-dependent manner. Survival was significantly increased in the TAT-PTEN9c treated group compared to saline controls at 2 h (14/15, 93% vs. 9/15, 60%, P < 0.05) and 4 h (10/15, 67% vs. 6/15, 40%, P < 0.05) after CPR. Treated mice had increased Akt phosphorylation within 30 min after CPR in heart and brain tissues with significantly decreased sorbitol content, suggesting improved metabolic recovery and glucose utilization. Conclusion: TAT-PTEN9c can be used after CPR in a mouse SCA model to rapidly enhance Akt activation and decrease glucose shunting via the polyol pathway in critical organs, preventing early cardiovascular collapse and death. Further work exploring the role of tissue sorbitol in sudden cardiac arrest injury is needed.

Introduction: Our previous work suggests that PBEF is an early blood marker inversely related to hypothermia protection and resuscitation survival in a mouse model of hemorrhagic shock. Given that neutrophils are a potential high-concentration source of blood PBEF, we studied mechanisms of PBEF release from isolated human neutrophils that could help design novel resuscitation strategies. Methods: Human blood neutrophils were isolated by Percoll density gradient. PBEF secretion into neutrophil supernatant was measured by ELISA. Results: Non-stimulated neutrophils (i.e. exposed to serum-starved conditions) released PBEF. PBEF in supernatant was observed at 5 min and plateaued after 30 min at 37oC. This release was not due to cell injury since LDH was unchanged, nor was it blocked by the classical secretion inhibitors, brefeldin A (blocks ER-Golgi transport) or nocodazole (blocks microtubule polymerization). As PBEF product NAD+ is consumed by multiple intra- and ecto-NADases, we determined whether inhibition of NADase activity impairs PBEF release. Nicotinamide, a global inhibitor of all NADases, blocked PBEF secretion by 73%, suggesting NADases promote PBEF secretion. To identify the specific NADase in PBEF secretion, neutrophils were treated with inhibitors for PARP, sirtuin, CD38 or mono-ADP-ribosyltransferase. We found that inhibition of CD38 by tannic acid or apigenin was able to block PBEF secretion in neutrophils (10.2 ± 2.8 ng/ml for control vs 1.8 ± 0.2 ng/ml for tannic acid at 100 μM, P < 0.01), but not by inhibitors of the other NADases. This observation indicated that CD38 glycohydroase activity is required for PBEF secretion. To further support the role of tannic acid in CD38 inhibition, tannic acid was shown to inhibit ε-ADP ribose production when neutrophils were treated with ε-NAD+. Finally, PBEF secretion was significantly enhanced by oxidative stress using H2O2 exposure. Conclusions: PBEF is constitutively released from neutrophils and this release is regulated by CD38 NAD glycohydrolase activity. Changes in human neutrophil microenvironment, especially oxidative stress, result in significant PBEF release within a timeframe that could account for the blood changes seen during mouse cardiovascular resuscitation.

In 12 anesthetized dogs, we tested the effects of pharmacologic beta-adrenergic stimulation on the severity of tracheal mast cell reactions in vivo (Am Rev Respir Dis 1979; 119:62). In control dogs...

Sudden cardiac arrest (SCA) is a leading cause of death in the United States. Despite return of spontaneous circulation, patients die due to post-SCA syndrome that includes myocardial dysfunction, brain injury, impaired metabolism, and inflammation. No medications improve SCA survival. Our prior work suggests that optimal Akt activation is critical for cooling protection and SCA recovery. Here, we investigate a small inhibitor of PTEN, an Akt-related phosphatase present in heart and brain, as a potential therapy in improving cardiac and neurological recovery after SCA. Anesthetized adult female wild-type C57BL/6 mice were randomized to pretreatment of VO-OHpic (VO) 30 min before SCA or vehicle control. Mice underwent 8 min of KCl-induced asystolic arrest followed by CPR. Resuscitated animals were hemodynamically monitored for 2 h and observed for 72 h. Outcomes included heart pressure-volume loops, energetics (phosphocreatine and ATP from 31P NMR), protein phosphorylation of Akt, GSK3β, pyruvate dehydrogenase (PDH) and phospholamban, circulating inflammatory cytokines, plasma lactate, and glucose as measures of systemic metabolic recovery. VO reduced deterioration of left ventricular maximum pressure, maximum rate of change in the left ventricular pressure, and Petco2 and improved 72 h neurological intact survival (50% vs. 10%; P < 0.05). It reduced plasma lactate, glucose, IL-1β, and Pre-B cell colony enhancing factor, while increasing IL-10. VO increased phosphorylation of Akt and GSK3β in both heart and brain, and cardiac phospholamban phosphorylation while reducing p-PDH. Moreover, VO improved cardiac bioenergetic recovery. We concluded that pharmacologic PTEN inhibition enhances Akt activation, improving metabolic, cardiovascular, and neurologic recovery with increased survival after SCA. PTEN inhibitors may be a novel pharmacologic strategy for treating SCA.

Reversible changes in lung microstructure accompany lung inflammation, although alterations in tissue micromechanics and their impact on inflammation remain unknown. This study investigated changes in extracellular matrix (ECM) remodeling and tissue stiffness in a model of LPS-induced inflammation and examined the role of lipoxin analog 15-epi-lipoxin A4 (eLXA4) in the reduction of stiffness-dependent exacerbation of the inflammatory process. Atomic force microscopy measurements of live lung slices were used to directly measure local tissue stiffness changes induced by intratracheal injection of LPS. Effects of LPS on ECM properties and inflammatory response were evaluated in an animal model of LPS-induced lung injury, live lung tissue slices, and pulmonary endothelial cell (EC) culture. In vivo, LPS increased perivascular stiffness in lung slices monitored by atomic force microscopy and stimulated expression of ECM proteins fibronectin, collagen I, and ECM crosslinker enzyme, lysyl oxidase. Increased stiffness and ECM remodeling escalated LPS-induced VCAM1 and ICAM1 expression and IL-8 production by lung ECs. Stiffness-dependent exacerbation of inflammatory signaling was confirmed in pulmonary ECs grown on substrates with high and low stiffness. eLXA4 inhibited LPS-increased stiffness in lung cross sections, attenuated stiffness-dependent enhancement of EC inflammatory activation, and restored lung compliance in vivo. This study shows that increased local vascular stiffness exacerbates lung inflammation. Attenuation of local stiffening of lung vasculature represents a novel mechanism of lipoxin antiinflammatory action.

Introduction: Our previous work suggests that PBEF, encoded by the NAMPT gene, is an early blood marker inversely related to hypothermia protection and resuscitation survival in a mouse model of hemorrhagic shock. Higher mouse blood PBEF concentrations were related to acute inflammation and cardiovascular collapse. More recently, clinical work by others demonstrated a correlation between high PBEF blood concentrations and acute heart failure exacerbation. Given that neutrophils are a potential high-concentration source of blood PBEF, we studied mechanisms of release from isolated human neutrophils that could help design novel resuscitation strategies. Methods: Human blood neutrophils were isolated by Percoll density gradient. PBEF secretion into neutrophil supernatant was measured by ELISA. Neutrophil PBEF protein expression was measured by Western blot analysis. Results: PBEF in unstimulated neutrophil supernatant was observed at 5 min and plateaued after 30 min at 37oC (8.6 ± 0.7 vs. 0.9 ± 0.3 ng/ml, p < 0.01). This release was not due to cell injury since LDH was unchanged, nor was it blocked by the classical secretion inhibitors, brefeldin A (blocks ER-Golgi transport) or nocodazole (blocks microtubule polymerization). However, PBEF secretion was attenuated by decreased temperature (4oC), suggesting a release mechanism other than passive diffusion. Indeed, glyburide an inhibitor of the ATP-binding cassette 1 (ABC 1) transporter reduced PBEF secretion by 55%. The calcium chelator EGTA completely blocked PBEF secretion. PBEF release was also dose-dependently blocked by extracellular ATP. Exogenous ATP induced the degradation of intracellular PBEF, which was blocked by pretreatment with a proteasomal inhibitor MG132, suggesting intracellular PBEF stability also regulates its secretion. Conclusions: PBEF is constitutively released from neutrophils via an unconventional secretion pathway. Its release is regulated by the ABC1 membrane transporter, calcium, and intracellular proteasome degradation. Changes in human neutrophil microenvironment result in significant change of PBEF release within a timeframe that could account for the blood changes seen during mouse cardiovascular resuscitation.

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-ΔPTEN p85) would enhance Akt phosphorylation to afford cardioprotection. Conversely, TAT fused p85 lacking the p110 binding site (TAT-Δp110p85) 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-Δp110 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-ΔPTEN 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.

The protective effects of prostacyclin and its stable analogue iloprost are mediated by elevation of intracellular cyclic AMP (cAMP) leading to enhancement of the peripheral actin cytoskeleton and cell–cell adhesive structures. This study tested the hypothesis that iloprost may exhibit protective effects against lung injury and endothelial barrier dysfunction induced by bacterial wall lipopolysaccharide (LPS).Endothelial barrier dysfunction was assessed by measurements of transendothelial permeability, morphologically and by analysis of LPS-activated inflammatory signalling.In vivo, C57BL/6J mice were challenged with LPS with or without iloprost or 8-bromoadenosine-3′,5′-cyclic monophosphate (Br-cAMP) treatment. Lung injury was monitored by measurements of bronchoalveolar lavage protein content, cell count and Evans blue extravasation.Iloprost and Br-cAMP attenuated the disruption of the endothelial monolayer, and suppressed the activation of p38 mitogen-activated protein kinase (MAPK), the nuclear factor (NF)-κB pathway, Rho signalling, intercellular adhesion molecular (ICAM)-1 expression and neutrophil migration after LPS challenge.In vivo, iloprost was effective against LPS-induced protein and neutrophil accumulation in bronchoalveolar lavage fluid, and reduced myeloperoxidase activation, ICAM-1 expression and Evans blue extravasation in the lungs. Inhibition of Rac activity abolished the barrier-protective and anti-inflammatory effects of iloprost and Br-cAMP.Iloprost-induced elevation of intracellular cAMP triggers Rac signalling, which attenuates LPS-induced NF-κB and p38 MAPK inflammatory pathways and the Rho-dependent mechanism of endothelial permeability.

Introduction: Cooling protects against cardiac ischemia/reperfusion (I/R) injury by enhancing PI3K/Akt/NOS signaling. PI3K/Akt signaling depends on binding by its regulatory subunit p85 to the catalytic subunit p110 or the inhibitory phosphatase PTEN. We studied novel TAT-p85 fusion proteins that selectively inhibit these competing p85 interactions. We hypothesized that TAT fused p85 lacking the PTEN binding site (TAT-ΔPTEN p85) would enhance Akt phosphorylation to afford cardioprotection in a heart cell model of I/R. Conversely, TAT fused p85 lacking the p110 binding site (TAT-Δp110p85) would abrogate protection. To further test TAT fusion proteins for the I/R injury of mouse cardiac arrest (CA), we studied whether a TAT-fused PTEN C-terminal 9 amino acid (TAT-PTEN9c) PTEN inhibitor would activate Akt in critical organs and improve survival. Methods: A TAT-Δp110 p85 plasmid was constructed with nucleotide deletions at amino acids 478-513. The TAT-ΔPTEN p85 plasmid deleted amino acids 1-313. TAT-PTEN9c was synthesized by Anaspec. Cardiomyocytes were isolated from 1-2-day old C57BL6/J mice and exposed to up to 90 min ischemic arrest with 3 h reperfusion (I/R). Cell viability was evaluated by propidium iodide uptake and p-Akt was measured by Western blot. C57BL6 mice were subjected to an established potassium-induced CA protocol. TAT-PTEN9c (7.8 mg/kg) was given IV after return of spontaneous circulation (ROSC) (n=10) and the 2nd dose for another 5 mice was given at 60 min after ROSC. Results: TAT-Δp110 p85 increased cell death from 44.6 +/- 2.7% to 92.5 +/- 3.4%, while TAT-ΔPTEN p85 decreased cell death caused by I/R from 47.8 +/- 1.4% to 11.9 +/- 5.3%. 30 mice were randomized to either saline or TAT-PTEN9c post-ROSC. TAT fusion proteins could be seen within heart and brain tissues within 2 minutes of IV infusion. The survival rate was significantly increased in TAT-PTEN9c treated mice compared with that of saline treated mice at 2 hrs (14/15, 93% vs. 9/15, 60%, P < 0.05) and at 4 hrs (10/15, 67% vs. 6/15, 40%, P < 0.05) after ROSC. Conclusions: TAT fusion protein inhibition of PTEN during cardiac I/R and after cardiac arrest appear to be promising strategies for enhancing Akt activation and survival.

BACKGROUND:Proline-rich tyrosine kinase 2 (Pyk2) is essential in neutrophil degranulation and chemotaxis in vitro. However, its effect on the process of lung inflammation and edema formation during LPS induced acute lung injury (ALI) remains unknown. The goal of the present study was to determine the effect of inhibiting Pyk2 on LPS-induced acute lung inflammation and injury in vivo.METHODS:C57BL6 mice were given either 10 mg/kg LPS or saline intratracheally. Inhibition of Pyk2 was effected by intraperitoneal administration TAT-Pyk2-CT 1 h before challenge. Bronchoalveolar lavage analysis of cell counts, lung histology and protein concentration in BAL were analyzed at 18 h after LPS treatment. KC and MIP-2 concentrations in BAL were measured by a mouse cytokine multiplex kit. The static lung compliance was determined by pressure-volume curve using a computer-controlled small animal ventilator. The extravasated Evans blue concentration in lung homogenate was determined spectrophotometrically.RESULTS:Intratracheal instillation of LPS induced significant neutrophil infiltration into the lung interstitium and alveolar space, which was attenuated by pre-treatment with TAT-Pyk2-CT. TAT-Pyk2-CT pretreatment also attenuated 1) myeloperoxidase content in lung tissues, 2) vascular leakage as measured by Evans blue dye extravasation in the lungs and the increase in protein concentration in bronchoalveolar lavage, and 3) the decrease in lung compliance. In each paradigm, treatment with control protein TAT-GFP had no blocking effect. By contrast, production of neutrophil chemokines MIP-2 and keratinocyte-derived chemokine in the bronchoalveolar lavage was not reduced by TAT-Pyk2-CT. Western blot analysis confirmed that tyrosine phosphorylation of Pyk2 in LPS-challenged lungs was reduced to control levels by TAT-Pyk2-CT pretreatment.CONCLUSIONS:These results suggest that Pyk2 plays an important role in the development of acute lung injury in mice and that pharmacological inhibition of Pyk2 might provide a potential therapeutic strategy in the pretreatment for patients at imminent risk of developing acute lung injury.