FXR protects lung from lipopolysaccharide-induced acute injury

Acute lung injury and its more severe form, acute respiratory distress syndrome, are characterized by an acute inflammatory response in the airspaces and lung parenchyma. The nuclear receptor farnesoid X receptor (FXR) is expressed in pulmonary artery endothelial cells. Here, we report a protective role of FXR in a lipopolysaccharide-induced mouse model of acute lung injury. Upon intratracheal injection of lipopolysaccharide, FXR-/- mice showed higher lung endothelial permeability, released more bronchoalveolar lavage cells to the alveoli, and developed acute pneumonia. Cell adhesion molecules were expressed at higher levels in FXR-/- mice as compared with control mice. Furthermore, lung regeneration was much slower in FXR-/- mice. In vitro experiments showed that FXR activation blocked TNFα-induced expression of P-selectin but stimulated proliferation of lung microvascular endothelial cells through up-regulation of Foxm1b. In addition, expression of a constitutively active FXR repressed the expression of proinflammatory genes and improved lung permeability and lung regeneration in FXR-/- mice. This study demonstrates a critical role of FXR in suppressing the inflammatory response in lung and promoting lung repair after injury.     

Influenza Infects Lung Microvascular Endothelium Leading to Microvascular Leak: Role of Apoptosis and Claudin-5

Severe influenza infections are complicated by acute lung injury, a syndrome of pulmonary microvascular leak. The pathogenesis of this complication is unclear. We hypothesized that human influenza could directly infect the lung microvascular endothelium, leading to loss of endothelial barrier function. We infected human lung microvascular endothelium with both clinical and laboratory strains of human influenza. Permeability of endothelial monolayers was assessed by spectrofluorimetry and by measurement of the transendothelial electrical resistance. We determined the molecular mechanisms of flu-induced endothelial permeability and developed a mouse model of severe influenza. We found that both clinical and laboratory strains of human influenza can infect and replicate in human pulmonary microvascular endothelium, leading to a marked increase in permeability. This was caused by apoptosis of the lung endothelium, since inhibition of caspases greatly attenuated influenza-induced endothelial leak. Remarkably, replication-deficient virus also caused a significant degree of endothelial permeability, despite displaying no cytotoxic effects to the endothelium. Instead, replication-deficient virus induced degradation of the tight junction protein claudin-5; the adherens junction protein VE-cadherin and the actin cytoskeleton were unaffected. Over-expression of claudin-5 was sufficient to prevent replication-deficient virus-induced permeability. The barrier-protective agent formoterol was able to markedly attenuate flu-induced leak in association with dose-dependent induction of claudin-5. Finally, mice infected with human influenza developed pulmonary edema that was abrogated by parenteral treatment with formoterol. Thus, we describe two distinct mechanisms by which human influenza can induce pulmonary microvascular leak. Our findings have implications for the pathogenesis and treatment of acute lung injury from severe influenza.     

In vitro effects of endotoxin on bovine and sheep lung microvascular and pulmonary artery endothelial cells

A single infusion of Escherichia coli endotoxin into sheep results in structural evidence of pulmonary endothelial injury, increases in both prostacyclin and prostaglandin E2 (PGE2) in lung lymph, and an increase in pulmonary microvascular permeability. Endotoxin-induced lung endothelial damage can also be induced in vitro, but to date these studies have utilized endothelium from large pulmonary vessels. In the present study, we have grown endothelial cells from peripheral lung vessels of cows and sheep and exposed these microvascular endothelial cells to endotoxin. Controls included lung microvascular endothelium without endotoxin and endothelial cells from bovine and sheep main pulmonary artery with and without addition of endotoxin. We found that endotoxin caused significant increases in release of prostacyclin and PGE2 from both bovine and sheep lung microvascular and pulmonary artery endothelium. Normal bovine and sheep pulmonary artery and bovine lung microvascular endothelium released greater levels of prostacyclin than PGE2 (ng/ng); release of PGE2 from the microvascular cells was greater than from the pulmonary artery endothelium in both species. Exposure of endothelial cells from cow and sheep main pulmonary artery to endotoxin results in endothelial cell retraction and pyknosis, a loss of barrier function, increased release of prostacyclin and PGE2 and eventual cell lysis. In lung microvascular cells, the increases in prostanoids were accompanied by changes in cell shape but occurred in the absence of either detectable alterations in barrier function or cytolysis. Thus, while endotoxin causes alterations to endothelial cells from both large and small pulmonary vessels, the effects are not identical suggesting site specific phenotypic expression of endothelial cells even within a single vessel. To determine whether the response of either the large or small pulmonary vessel endothelial cells in culture mimics most closely the in vivo response of the lung to endotoxin requires further study.     

Lycium barbarum polysaccharide protects against LPS-induced ARDS by inhibiting apoptosis,oxidative stress,and inflammation in pulmonary endothelial cells

Acute respiratory distress syndrome (ARDS) is a heterogenous syndrome characterised by diffuse alveolar damage, with an increase in lung endothelial and epithelial permeability. Lycium barbarum polysaccharide (LBP), the most biologically active fraction of wolfberry, possesses antiapoptotic and antioxidative effects in distinct situations. In the present study, the protective effects and potential molecular mechanisms of LBP against lipopolysaccharide (LPS)-induced ARDS were investigated in the mice and in the human pulmonary microvascular endothelial cells (HPMECs). The data indicated that pretreatment with LBP significantly attenuated LPS-induced lung inflammation and pulmonary oedema in vivo. LBP significantly reversed LPS-induced decrease in cell viability, increase in apoptosis and oxidative stress via inhibiting caspase-3 activation and intracellular reactive oxygen species (ROS) production in vitro. Moreover, the scratch assay verified that LBP restored the dysfunction of endothelial cells (ECs) migration induced by LPS stimulation. Furthermore, LBP also significantly suppressed LPS-induced NF-κB activation, and subsequently reversed the release of cytochrome c. These results showed the antiapoptosis and antioxidant LBP could partially protect against LPS-induced ARDS through promoting the ECs survival and scavenging ROS via inhibition of NF-κB signalling pathway. Thus, LBP could be potentially used for ARDS against pulmonary inflammation and pulmonary oedema.     

Role of CPI-17 in the regulation of endothelial cytoskeleton

We have previously shown that myosin light chain (MLC) phosphatase (MLCP) is critically involved in the regulation of agonist-mediated endothelial permeability and cytoskeletal organization (Verin AD, Patterson CE, Day MA, and Garcia JG. Am J Physiol Lung Cell Mol Physiol 269: L99-L108, 1995). The molecular mechanisms of endothelial MLCP regulation, however, are not completely understood. In this study we found that, similar to smooth muscle, lung microvascular endothelial cells expressed specific endogenous inhibitor of MLCP, CPI-17. To elucidate the role of CPI-17 in the regulation of endothelial cytoskeleton, full-length CPI-17 plasmid was transiently transfected into pulmonary artery endothelial cells, where the background of endogenous protein is low. CPI-17 had no effect on cytoskeleton under nonstimulating conditions. However, stimulation of transfected cells with direct PKC activator PMA caused a dramatic increase in F-actin stress fibers, focal adhesions, and MLC phosphorylation compared with untransfected cells. Inflammatory agonist histamine and, to a much lesser extent, thrombin were capable of activating CPI-17. Histamine caused stronger CPI-17 phosphorylation than thrombin. Inhibitory analysis revealed that PKC more significantly contributes to agonist-induced CPI-17 phosphorylation than Rho-kinase. Dominant-negative PKC-alpha abolished the effect of CPI-17 on actin cytoskeleton, suggesting that the PKC-alpha isoform is most likely responsible for CPI-17 activation in the endothelium. Depletion of endogenous CPI-17 in lung microvascular endothelial cell significantly attenuated histamine-induced increase in endothelial permeability. Together these data suggest the potential importance of PKC/CPI-17-mediated pathway in histamine-triggered cytoskeletal rearrangements leading to lung microvascular barrier compromise.     

Rho inhibition decreases TNF-induced endothelial MAPK activation and monolayer permeability.

Our laboratory previously demonstrated that MAPK activation is an important signal during cytokine-induced endothelial permeability (Nwariaku FE, Liu Z, Terada L, Duffy S, Sarosi G, and Turnage R. Shock 18: 82-85, 2002). Because GTP-binding proteins have been implicated in MAPK activation, we now hypothesize that the GTP-binding protein Rho is a mediator of TNF-induced MAPK activation and increased endothelial permeability. Transmonolayer permeability was assessed in human lung microvascular cells by measuring transmonolayer electrical resistance. MAPK activity was assessed by using a phospho-specific immunoprecipitation kinase assay and by comparing Western blots for phospho-MAPK with total MAPK. MAPK inhibitors used were SB-202190 and PD-098059, whereas Clostridium botulinum C3 transferase was used as a Rho inactivator. Rho-associated coiled-coil kinase was inhibited with Y-27632. TNF increased pulmonary endothelial permeability in vitro and caused a rapid, sustained increase in endothelial p38 and extracellular signal-regulated kinase MAPK activity. Inhibition of p38 and extracellular signal-regulated kinase MAPK with SB-202190 and PD-098059, respectively, decreased TNF-induced endothelial permeability. C3 transferase attenuated TNF-induced MAPK activation and blocked TNF-induced endothelial permeability. Finally, inhibition of Rho-associated coiled-coil kinase with Y-27632 prevented both MAPK activation and TNF-induced decreases in transmonolayer resistance. Rho acts upstream of mitogen-activated protein kinases in mediating TNF-induced pulmonary endothelial leak.     

Redox regulation of endothelial barrier integrity

Intestinal ischemia-reperfusion is associated with the generation of reactive oxygen metabolites as well as remote, oxidant-mediated lung injury. Oxidants elicit endothelial redox imbalance and loss of vascular integrity by disorganizing several junctional proteins that contribute to the maintenance and regulation of the endothelial barrier. To determine the specific effect of redox imbalance on pulmonary vascular barrier integrity, microvascular permeability was determined in lungs of animals subjected to chemically induced redox imbalance. The effect of redox imbalance on microvascular permeability and endothelial junctional integrity in cultured lung microvascular cells was also determined. Whole lung and cultured pulmonary endothelial cell permeability both increased significantly in response to chemical redox imbalance. Thiol depletion also resulted in decreased endothelial cadherin content and disruption of the endothelial barrier. These deleterious effects of intracellular redox imbalance were blocked by pretreatment with exogenous glutathione. The results of this study suggest that redox imbalance contributes to pulmonary microvascular dysfunction by altering the content and/or spatial distribution of endothelial junctional proteins.     

蛋白激酶在微血管通透性中的作用

微血管内皮细胞层是一层半选择通透性屏障,可以调节血液中的液体、溶质和血浆蛋白进入组织间隙。在炎症刺激作用下,可通过旁细胞途径和跨细胞途径引起内皮通透性上升。旁细胞通路主要由内皮细胞间的紧密连接、黏附连接和细胞与外基质的黏着斑组成。炎症介质,如脂多糖和肿瘤坏死因子α可激活多种蛋白激酶。活化的蛋白激酶主要包括Rho相关的卷曲蛋白激酶、肌球蛋白轻链激酶、蛋白激酶C、酪氨酸激酶和丝裂原活化蛋白激酶等,参与引发内皮屏障生化和结构改变,旁细胞通路开放,导致通透性上升。该文对上述蛋白激酶在微血管通透性中作用机制的研究进展进行综述。     

Role of Rho-kinase in reexpansion pulmonary edema in rabbits

Reexpansion of a collapsed lung increases the microvascular permeability and causes reexpansion pulmonary edema. Neutrophils and their products have been implicated in the development of this phenomenon. The small GTP-binding proteins Rho and its target Rho-kinase (ROCK) regulate endothelial permeability, although their roles in reexpansion pulmonary edema remain unclear. We studied the contribution of ROCK to pulmonary endothelial and epithelial permeability in a rabbit model of this disorder. Endothelial and epithelial permeability was assessed by measuring the tissue-to-plasma (T/P) and bronchoalveolar lavage (BAL) fluid-to-plasma (B/P) ratios with (125)I-labeled albumin. After intratracheal instillation of (125)I-albumin, epithelial permeability was also assessed from the plasma leak (PL) index, the ratio of (125)I-albumin in plasma/total amount of instilled (125)I-albumin. T/P, B/P, and PL index were significantly increased in the reexpanded lung. These increases were attenuated by pretreatment with Y-27632, a specific ROCK inhibitor. However, neutrophil influx, neutrophil elastase activity, and malondialdehyde concentrations in BAL fluid collected from the reexpanded lung were not changed by Y-27632. In endothelial monolayers, Y-27632 significantly attenuated the H(2)O(2)-induced increase in permeability and mitigated the morphological changes in the actin microfilament cytoskeleton of endothelial cells. These in vivo and in vitro observations suggest that the Rho/ROCK pathway contributes to the increase in alveolar barrier permeability associated with reexpansion pulmonary edema.     

Induced Pluripotent Stem Cell Therapy Ameliorates Hyperoxia-Augmented Ventilator-Induced Lung Injury through Suppressing the Src Pathway

Background

High tidal volume (V_T) mechanical ventilation (MV) can induce the recruitment of neutrophils, release of inflammatory cytokines and free radicals, and disruption of alveolar epithelial and endothelial barriers. It is proposed to be the triggering factor that initiates ventilator-induced lung injury (VILI) and concomitant hyperoxia further aggravates the progression of VILI. The Src protein tyrosine kinase (PTK) family is one of the most critical families to intracellular signal transduction related to acute inflammatory responses. The anti-inflammatory abilities of induced pluripotent stem cells (iPSCs) have been shown to improve acute lung injuries (ALIs); however, the mechanisms regulating the interactions between MV, hyperoxia, and iPSCs have not been fully elucidated. In this study, we hypothesize that Src PTK plays a critical role in the regulation of oxidants and inflammation-induced VILI during hyperoxia. iPSC therapy can ameliorate acute hyperoxic VILI by suppressing the Src pathway.

Methods

Male C57BL/6 mice, either wild-type or Src-deficient, aged between 2 and 3 months were exposed to high V_T (30 mL/kg) ventilation with or without hyperoxia for 1 to 4 h after the administration of Oct4/Sox2/Parp1 iPSCs at a dose of 5×10⁷ cells/kg of mouse. Nonventilated mice were used for the control groups.

Results

High V_T ventilation during hyperoxia further aggravated VILI, as demonstrated by the increases in microvascular permeability, neutrophil infiltration, macrophage inflammatory protein-2 (MIP-2) and plasminogen activator inhibitor-1 (PAI-1) production, Src activation, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, and malaldehyde (MDA) level. Administering iPSCs attenuated ALI induced by MV during hyperoxia, which benefited from the suppression of Src activation, oxidative stress, acute inflammation, and apoptosis, as indicated by the Src-deficient mice.

Conclusion

The data suggest that iPSC-based therapy is capable of partially suppressing acute inflammatory and oxidant responses that occur during hyperoxia-augmented VILI through the inhibition of Src-dependent signaling pathway.     

Mechanisms of lung endothelial barrier disruption induced by cigarette smoke: role of oxidative stress and ceramides

The epithelial and endothelial cells lining the alveolus form a barrier essential for the preservation of the lung respiratory function, which is, however, vulnerable to excessive oxidative, inflammatory, and apoptotic insults. Whereas profound breaches in this barrier function cause pulmonary edema, more subtle changes may contribute to inflammation. The mechanisms by which cigarette smoke (CS) exposure induce lung inflammation are not fully understood, but an early alteration in the epithelial barrier function has been documented. We sought to investigate the occurrence and mechanisms by which soluble components of mainstream CS disrupt the lung endothelial cell barrier function. Using cultured primary rat microvascular cell monolayers, we report that CS induces endothelial cell barrier disruption in a dose- and time-dependent manner of similar magnitude to that of the epithelial cell barrier. CS exposure triggered a mechanism of neutral sphingomyelinase-mediated ceramide upregulation and p38 MAPK and JNK activation that were oxidative stress dependent and that, along with Rho kinase activation, mediated the endothelial barrier dysfunction. The morphological changes in endothelial cell monolayers induced by CS included actin cytoskeletal rearrangement, junctional protein zonula occludens-1 loss, and intercellular gap formation, which were abolished by the glutathione modulator N-acetylcysteine and ameliorated by neutral sphingomyelinase inhibition. The direct application of ceramide recapitulated the effects of CS, by disrupting both endothelial and epithelial cells barrier, by a mechanism that was redox and apoptosis independent and required Rho kinase activation. Furthermore, ceramide induced dose-dependent alterations of alveolar microcirculatory barrier in vivo, measured by two-photon excitation microscopy in the intact rat. In conclusion, soluble components of CS have direct endothelial barrier-disruptive effects that could be ameliorated by glutathione modulators or by inhibitors of neutral sphingomyelinase, p38 MAPK, JNK, and Rho kinase. Amelioration of endothelial permeability may alleviate lung and systemic vascular dysfunction associated with smoking-related chronic obstructive lung diseases.     

Regulatory role for nucleosome assembly protein-1 in the proliferative and vasculogenic phenotype of pulmonary endothelium

Mediators of angiogenesis such as VEGFs and angiopoietins may regulate pulmonary vascular permeability under normal and pathological conditions. Ephrin family receptor tyrosine kinases are expressed in the vasculature and also regulate angiogenesis under some circumstances, but whether they also modulate lung vascular permeability is unknown. We hypothesized that stimulation of lung endothelial EphA receptors with ephrin-a1 ligand would alter pulmonary vascular permeability and tested this idea in vivo and in vitro. We found that ephrin-a1 ligand and EphA2 receptors are expressed in distal normal lung vasculature and that their expression is increased in injured lung, suggesting a link to mechanisms of increased permeability. Intravenous injection of ephrin-a1 caused a large increase in the leakage of labeled albumin into the lungs of rats within 30 min (293 +/- 27 vs. 150 +/- 6 ng/mg dry lung, P < 0.01), along with histological evidence of the formation of endothelial disruptions. In cultured lung vascular endothelial cells, stimulation with ephrin-a1 increased monolayer permeability by 44% (P < 0.01), a permeability change similar to that seen with VEGF stimulation of the same cells. Ephrin-a1 stimulation in vivo and in vitro was associated with histological evidence for disruptions of tight and adherens junctions. These observations describe a novel role for ephrin-a1 and EphA receptors in the regulation of vascular permeability in the lung.     

LPS induces permeability injury in lung microvascular endothelium via AT(1) receptor

Lipopolysaccharide (LPS) is known to stimulate the circulation and local production of angiotensin II (Ang II). To assess whether Ang II plays a role in LPS-induced acute lung injury, rats were injected with LPS, the microvascular endothelial permeability injury was evaluated by histological changes, increased pulmonary wet/dry weight ratio, and pulmonary microvascular protein leak. Besides, increased rat pulmonary microvascular endothelial cell monolayer permeability coefficient (K(f)) was measured after treatment with LPS and/or Ang II, respectively. LPS/Ang II, treatment resulted in a significant increase in K(f). Ang II cooperates with LPS to further increase K(f). Hence, LPS increases pulmonary microvascular endothelial permeability both in vitro and in vivo. Local lung Ang II was increased in response to LPS challenge, and elevated Ang II ulteriorly exacerbates LPS-induced endothelium injury. [Sar(1),Ile(8)]Ang II, a selective block of Ang II type 1 (AT(1)) receptors, eliminated these changes significantly. Our conclusion is that the LPS-induced lung injury may be mediated by the AT(1) receptor.     

Mechanical stress activates xanthine oxidoreductase through MAP kinase-dependent pathways

Xanthine oxidoreductase (XOR) plays a prominent role in acute lung injury because of its ability to generate reactive oxygen species. We investigated the role of XOR in ventilator-induced lung injury (VILI). Male C57BL/6J mice were assigned to spontaneous ventilation (sham) or mechanical ventilation (MV) with low (7 ml/kg) and high tidal volume (20 ml/kg) for 2 h after which lung XOR activity and expression were measured and the effect of the specific XOR inhibitor allopurinol on pulmonary vascular leakage was examined. In separate experiments, rat pulmonary microvascular endothelial cells (RPMECs) were exposed to cyclic stretch (5% and 18% elongation, 20 cycles/min) for 2 h before intracellular XOR activity measurement. Lung XOR activity was significantly increased at 2 h of MV without changes in XOR expression. There was evidence of p38 MAP kinase, ERK1/2, and ERK5 phosphorylation, but no change in JNK phosphorylation. Evans blue dye extravasation and bronchoalveolar lavage protein concentration were significantly increased in response to MV, changes that were significantly attenuated by pretreatment with allopurinol. Cyclic stretch of RPMECs also caused MAP kinase phosphorylation and a 1.7-fold increase in XOR activity, which was completely abrogated by pretreatment of the cells with specific MAP kinase inhibitors. We conclude that XOR enzymatic activity is significantly increased by mechanical stress via activation of p38 MAP kinase and ERK and plays a critical role in the pathogenesis of pulmonary edema associated with VILI.     

Protein kinase C-mediated endothelial barrier regulation is caveolin-1-dependent

Protein kinase C (PKC) is activated in response to various inflammatory mediators and contributes significantly to the endothelial barrier breakdown. However, the mechanisms underlying PKC-mediated permeability regulation are not well understood. We prepared microvascular myocardial endothelial cells from both wild-type (WT) and caveolin-1-deficient mice. Activation of PKC by phorbol myristate acetate (PMA) (100 nM) for 30 min induced intercellular gap formation and fragmentation of VE-cadherin immunoreactivity in WT but not in caveolin-1-deficient monolayers. To test the effect of PKC activation on VE-cadherin-mediated adhesion, we allowed VE-cadherin-coated microbeads to bind to the endothelial cell surface and probed their adhesion by laser tweezers. PMA significantly reduced bead binding to 78±6% of controls in WT endothelial cells without any effect in caveolin-1-deficient cells. In WT cells, PMA caused an 86±18% increase in FITC-dextran permeability whereas no increase in permeability was observed in caveolin-1-deficient monolayers. Inhibition of PKC by staurosporine (50 nM, 30 min) did not affect barrier functions in both WT and caveolin-1-deficient MyEnd cells. Theses data indicate that PKC activation reduces endothelial barrier functions at least in part by the reduction of VE-cadherin-mediated adhesion and demonstrate that PKC-mediated permeability regulation depends on caveolin-1.     

Zymosan-activated plasma causes prolonged decreases in PMN superoxide release in sheep

It is well established that activation of neutrophils within the pulmonary circulation produces acute lung injury in which adherence of neutrophils to endothelial cells is an obligatory step in the mechanism of injury. The effects of in vivo activation of neutrophils on the in vitro responses of these cells to stimulation have not been determined, although such information may be important in understanding how different etiological factors may interact to produce infection or acute respiratory failure. By using an assay to sequentially measure superoxide anion (O2-) release from adherent neutrophils stimulated with phorbol myristate acetate (PMA), we measured the in vitro activation response of peripheral blood neutrophils isolated before and 24 h after infusion of zymosan-activated plasma (ZAP; or untreated plasma as a control), air bubbles, or PMA in awake, instrumented sheep. Each of the three inflammatory agents produced an increase in lung microvascular permeability characteristic of acute lung injury; control plasma did not. For the in vivo ZAP experiments, stimulated O2- release in vitro by using PMA was approximately 50% lower (P less than 0.05) for neutrophils isolated 24 h after the in vivo infusion (4.3 +/- 0.8 nmol/500,000 cells) than before (8.1 +/- 0.2 nmol/500,000 cells). For the air emboli or PMA in vivo experiments, there were no changes in neutrophil activation responses in vitro. Similarly, infusion of control plasma did not result in reduced neutrophil O2- release. These results show that alterations in the inflammatory potential of neutrophils may occur in vivo and that such alterations appear to be dependent on the mechanism and agent by which lung injury is produced.     

RhoA and Rho-kinase dependent and independent signals mediate TGF-beta-induced pulmonary endothelial cytoskeletal reorganization and permeability

Transforming growth factor (TGF)-beta is a potent inflammatory mediator involved in acute lung injury. TGF-beta directly increases pulmonary endothelial myosin light chain (MLC) phosphorylation, which is associated with increased endothelial stress fiber formation, gap formation, and protein permeability, all hallmarks of pulmonary endothelial responses during acute lung injury. We performed the following experiments in pulmonary endothelial monolayers to determine whether RhoA and Rho-kinase mediate these TGF-beta-induced responses. TGF-beta caused the sustained activation of RhoA 2 h posttreatment associated with increased MLC phosphorylation. Inhibition of either RhoA or Rho-kinase with either C3 exoenzyme or Y-27632 blocked MLC phosphorylation. In addition, both C3 and Y-27632 partially attenuated the maximal TGF-beta-induced increase in permeability but did not affect the initial phase of compromised barrier integrity. Inhibition of Rho-kinase completely blocked the TGF-beta-induced increase in the content of filamentous actin (F-actin) but only partially inhibited TGF-beta-induced changes in actin reorganization. To assess the contribution of Rho-kinase in RhoA-mediated responses independent of additional TGF-beta-induced signals, cells were infected with a constitutively active RhoA adenovirus (RhoAQ63L) with or without Y-27632. RhoAQ63L increased MLC phosphorylation, F-actin content, and permeability. Treatment with Y-27632 blocked these responses, suggesting that Rho-kinase mediates these RhoA-induced effects. Collectively, these data suggest the following: 1) the RhoA/Rho-kinase pathway is an important component of TGF-beta-induced effects on endothelial MLC phosphorylation, cytoskeletal reorganization, and barrier integrity; and 2) additional signaling mechanisms independent of the RhoA/Rho-kinase signaling cascade contribute to TGF-beta-induced changes in cytoskeletal organization and permeability.     

Losartan attenuates microvascular permeability in mechanical ventilator-induced lung injury in diabetic mice

Mechanical ventilation can cause direct injury to the lungs, a type of injury known as ventilator-induced lung injury (VILI). VILI is associated with up-regulates angiotensinogen and AT1 receptor expression of in the lung. This work explored effects of losartan on VILI in diabetic mice. Ninty-six C57Bl/6 mice were randomly divided into six groups, control group (C group), diabetes group (D group), diabetes mechanical ventilation group (DV group), losartan control group (L + C group), losartan treatment group in diabetic mice (L + D group) and losartan treatment group in mechanical ventilation diabetic mice (L + DV group). Lung W/D, myeloperoxidase (MPO) activity, microvascular permeability, blood–gas analysis, Ang II concentrations and AT-1R protein expression were measured. Compared with D group, DV group increased Ang II concentrations, AT-1R protein expression, W/D ratio, MPO activity, and microvascular permeability. PaO₂ were significantly lower in the DV group than D group or control group. Compared with DV group, L + DV group attenuates ventilator-induced lung injury in diabetic mice and prevented the increase Ang II concentrations, AT-1R protein expression and microvascular permeability caused by ventilation in diabetic mice. This study provides in vivo evidence that losartan attenuates microvascular permeability via down-regulates Ang II and AT-1R expression in mechanical ventilator-induced lung injury in diabetic mice.     

Deletion of myosin light chain kinase in endothelial cells has a minor effect on the lipopolysaccharide-induced increase in microvascular endothelium permeability in mice

There is a current view that myosin light chain kinase (MLCK) plays a critical role in endothelial permeability. To investigate the functions of MLCK in endothelial cells in vivo, we generated a mouse model in which MLCK was selectively deleted by crossing Mylk1 floxed mice with Tie2/cre transgenic mice. Knocking out Mylk1 from endothelial cells had no effect on the global phenotype of the mice, including body weight and blood pressure. Lipopolysaccharide (LPS)-mediated septic death was also not altered in the knockout (KO) mice. Consistently, LPS-induced inflammatory injury and the increase in microvascular permeability in the main organs, including the lung and the kidney, was not significantly attenuated in KO mice as compared with wild-type mice. However, the LPS-induced microvascular hyperpermeability of the esophagus and the eyeballs was attenuated in KO mice. We also found that the LPS-mediated increase in the number of caveolae in the endothelial cells of the esophagus was significantly reduced in KO mice. Our results do not support a role for endothelial cell MLCK in the pathogenesis of inflammatory diseases.     

Mechanisms of early pulmonary neutrophil sequestration in ventilator-induced lung injury in mice

Polymorphonuclear leukocytes (PMN) play an important role in ventilator-induced lung injury (VILI), but the mechanisms of pulmonary PMN recruitment, particularly early intravascular PMN sequestration during VILI, have not been elucidated. We investigated the physiological and molecular mechanisms of pulmonary PMN sequestration in an in vivo mouse model of VILI. Anesthetized C57/BL6 mice were ventilated for 1 h with high tidal volume (injurious ventilation), low tidal volume and high positive end-expiratory pressure (protective ventilation), or normal tidal volume (control ventilation). Pulmonary PMN sequestration analyzed by flow cytometry of lung cell suspensions was substantially enhanced in injurious ventilation compared with protective and control ventilation, preceding development of physiological signs of lung injury. Anesthetized, spontaneously breathing mice with continuous positive airway pressure demonstrated that raised alveolar pressure alone does not induce PMN entrapment. In vitro leukocyte deformability assay indicated stiffening of circulating leukocytes in injurious ventilation compared with control ventilation. PMN sequestration in injurious ventilation was markedly inhibited by administration of anti-L-selectin antibody, but not by anti-CD18 antibody. These results suggest that mechanical ventilatory stress initiates pulmonary PMN sequestration early in the course of VILI, and this phenomenon is associated with stretch-induced inflammatory events leading to PMN stiffening and mediated by L-selectin-dependent but CD18-independent mechanisms.