Pulmonary reexpansion causes xanthine oxidase-induced apoptosis in rat lung

The pathogenesis of reexpansion pulmonary edema is not yet fully understood. We therefore studied its mechanism in a rat model in which the left lung was collapsed by bronchial occlusion for 1 h and then reexpanded and ventilated for an additional 3 h. We then evaluated the production of reactive oxygen species in the lungs using fluorescent imaging and cerium deposition electron microscopic techniques and the incidence of apoptosis using the TdT-mediated dUTP-digoxigenin nick end labeling (TUNEL) method. We found that pulmonary reexpansion induced production of reactive oxygen species and then apoptosis, mainly in endothelial and alveolar type II epithelial cells. Endothelial cells and alveolar type I and II epithelial cells in the reexpanded lung were positive for TUNEL and cleaved caspase-3. DNA fragmentation was also observed in the reexpanded lung. In addition, wet-dry ratios obtained with reexpanded lungs were significantly higher than those obtained with control lungs, indicating increased fluid content. All of these effects were attenuated by pretreating rats with a specific xanthine oxidase inhibitor, sodium (-)-8-(3-methoxy-4-phenylsulfinylphenyl) pyrazolo[1,5-a]-1,3,5-triazine-4(1H)-one. It thus appears that pulmonary reexpansion activates xanthine oxidase in both endothelial and alveolar type II epithelial cells and that the reactive oxygen species produced by the enzyme induce apoptosis among the endothelial and alveolar type I and II epithelial cells that make up the pulmonary water-air barrier, leading to reexpansion pulmonary edema.     

Neutrophils in reexpansion pulmonary edema

This study investigated the possible contribution of neutrophils to development of reexpansion pulmonary edema (RPE) in rabbits. Rabbits' right lungs were collapsed for 7 days and then reexpanded with negative intrathoracic pressure for 2 h before study, a model that creates unilateral edema in the reexpanded lungs but not in contralateral left lungs. Two hours after lung reexpansion, significant increases in lavage albumin concentration (17-fold), percent neutrophils (14-fold), and total number of neutrophils (7-fold) recovered occurred in the reexpanded lung but not in the left. After 2 h of reexpansion increased leukotriene B4 was detected in lavage supernatant from right lungs (335 +/- 33 pg/ml) compared with the left (110 +/- 12 pg/mg, P less than 0.01), and right lung lavage acid phosphatase activity similarly increased (6.67 +/- 0.35 U/l) compared with left (4.73 +/- 0.60 U/l, P less than 0.05). Neutropenia induced by nitrogen mustard (17 +/- 14 greater than neutrophils/microliters) did not prevent RPE, because reexpanded lungs from six neutropenic rabbits were edematous (wet-to-dry lung weight ratio 6.34 +/- 0.43) compared with their contralateral lungs (4.97 +/- 0.04, P less than 0.01). An elevated albumin concentration in reexpanded lung lavage from neutropenic rabbits (8-fold) confirmed an increase in permeability. Neutrophil depletion before reexpansion did not prevent unilateral edema, although neutrophils were absent from lung sections and alveolar lavage fluid from neutropenic rabbits.     

ROCK mediates thrombin's endothelial barrier dysfunction

Thrombin-induced endothelial monolayer hyperpermeability is thought toresult from increased F-actin stress fiber-related contractile tension,a process regulated by the small GTP-binding protein Rho. We testedwhether this process was dependent on the Rho-associated proteinkinase, ROCK, using a specific ROCK inhibitor, Y-27632. The effects ofY-27632 on thrombin-induced myosin light chain phosphorylation (MLCP)and tyrosine phosphorylation of p125 focal adhesion kinase(p125^FAK) and paxillin were measured by Western blotting.F-actin organization and content were analyzed by digital imaging, andendothelial monolayer permeability was measured in bovine pulmonaryartery endothelial cell (EC) monolayers using a size-selectivepermeability assay. Y-27632 enhanced EC monolayer barrier function dueto a decline in small-pore number that was associated with increased ECsurface area, reduced F-actin content, and reorganization of F-actin to-catenin-containing cell-cell adherens junctions. Although Y-27632prevented thrombin-induced MLCP, stress fiber formation, and theincreased phosphotyrosine content of paxillin and p125^FAK,it attenuated but did not prevent the thrombin-induced formation oflarge paracellular holes. These data indicate that thrombin-induced stress fiber formation is ROCK dependent. In contrast, thrombin-induced paracellular hole formation occurs in a ROCK-independent manner, whereas thrombin-induced monolayer hyperpermeability appears to bepartially ROCK dependent.

     

Rho kinases regulate corneal epithelial wound healing

We have previously shown that Rho small GTPase is required for modulating both cell migration and proliferation through cytoskeleton reorganization and focal adhesion formation in response to wounding. In the present study, we investigated the role of Rho kinases (ROCKs), major effectors of Rho GTPase, in mediating corneal epithelial wound healing. Both ROCK 1 and 2 were expressed and activated in THCE cells, an SV40-immortalized human corneal epithelial cell (HCEC) line, in response to wounding, lysophosphatidic acid, and heparin-binding EGF-like growth factor (HB-EGF) stimulations. The ROCK inhibitor Y-27632 efficiently antagonized ROCK activities without affecting Rho activation in wounded HCECs. Y-27632 promoted basal and HB-EGF-enhanced scratch wound healing and enhanced cell migration and adhesion to matrices, while retarded HB-EGF induced cell proliferation. E-cadherin- and beta-catenin-mediated cell-cell junction and actin cytoskeleton organization were disrupted by Y-27632. Y-27632 impaired the formation and maintenance of tight junction barriers indicated by decreased trans-epithelial resistance and disrupted occludin staining. We conclude that ROCK activities enhance cell proliferation, promote epithelial differentiation, but negatively modulate cell migration and cell adhesion and therefore play a role in regulating corneal epithelial wound healing.     

Chronic intrauterine pulmonary hypertension increases endothelial cell Rho kinase activity and impairs angiogenesis in vitro

Persistent pulmonary hypertension of the newborn (PPHN) is characterized by endothelial dysfunction and decreased vascular growth. The role of Rho kinase activity in modulating endothelial function and regulating angiogenesis during normal lung development and in PPHN is unknown. We hypothesized that PPHN increases Rho kinase activity in fetal pulmonary artery endothelial cells (PAECs) and impairs angiogenesis in vitro. Proximal PAECs were harvested from fetal sheep with partial ligation of the ductus arteriosus in utero (PPHN) and age-matched controls. Rho kinase activity was measured by RhoA, Rho GTP, and phosphorylated MYPT-1 protein content. The effects of Rho kinase activity on angiogenesis, endothelial nitric oxide (NO) synthase (eNOS) protein expression, and NO production were determined in normal and PPHN PAECs. Angiogenesis was assessed by tube formation in vitro with/without Y-27632 (a Rho kinase inhibitor) and calpeptin (a Rho kinase activator) in the presence/absence of N-nitro-l-arginine (l-NA, an NOS inhibitor). RhoA, Rho GTP, and phosphorylated MYPT-1 protein were increased in PPHN PAECs. Tube formation was reduced 29% in PPHN PAECs (P < 0.001) and increased with Y-27632 treatment in normal and PPHN PAECs, with PPHN PAECs achieving levels similar to those of normal PAECs. l-NA inhibited the Y-27632-induced increase in tube formation in normal, but not PPHN, PAECs. Calpeptin reduced tube formation in normal and PPHN PAECs. eNOS expression was reduced 42% in PPHN PAECs (P < 0.01). Y-27632 increased eNOS protein and NO production in normal and PPHN PAECs. Calpeptin decreased eNOS protein only in normal PAECs but reduced NO production in normal and PPHN PAECs. We conclude that Rho kinase activity is increased in PPHN PAECs and impairs angiogenesis and downregulates eNOS protein and NO production in vitro.     

Endogenous and exogenous catalase in reoxygenation lung injury.

Reexpansion pulmonary edema parallels reperfusion (reoxygenation) injuries in other organs in that hypoxic and hypoperfused lung tissue develops increased vascular permeability and neutrophil infiltration after reexpansion. This study investigated endogenous lung catalase activity and H2O2 production during hypoxia (produced by lung collapse) and after reoxygenation (resulting from reexpansion), in addition to assessing the effects of exogenous catalase infusion on the development of unilateral pulmonary edema after reexpansion. Lung collapse resulted in a progressive increase in endogenous catalase activity after 3 (14%) and 7 days (23%), while activities in contralateral left lungs did not change (normal left lungs averaged 180 +/- 11 units/mg DNA). Tissue from control left lungs released H2O2 into the extracellular medium at a rate calculated to be 242 +/- 34 nmol.h-1.lung-1. No significant change in extracellular release of H2O2 occurred after 7 days of right lung collapse. However, after reexpansion of the previously collapsed right lungs for 2 h, H2O2 release from both reexpanded right and contralateral left lungs significantly increased (88 and 60%, respectively) compared with controls. Infusion of exogenous catalase significantly increased plasma and lung catalase activities. Exogenous catalase infusion prevented neither the increase in lung permeability nor the infiltration with neutrophils that typically occurs in reexpanded lungs. These data indicate that lung hypoxia/reoxygenation, induced by sequential collapse and reexpansion, has specific effects on endogenous lung catalase activity and H2O2 release. However, exogenous catalase does not prevent reexpansion pulmonary edema, eliminating extracellular (but not intracellular) H2O2 as an important mediator of unilateral lung injury in this model.     

Solute permeability of the alveolar epithelium in alloxan edema in dogs

The permeability of the alveolar epithelium following alloxan challenge was studied in dogs by determining transfer of radiolabeled solutes between alveolus and blood. Two days after injection of 131-Ialbumin into the blood, anesthetized dogs had the air space of part of one lung isolated by a balloon catheter lodged in a bronchus. We infused the atelectatis-isolated area with normal saline containing trace amounts of Blue Dextran, 125Ialbumin, and 57Co-cyanocobalamin; challenged six animals with intravenous alloxan, and six animals with alloxan added to the alveolar saline. During the pulmonary edema, 57Co-cyanocabalamin and 125I-albumin appeared in the blood and 131I-albumin entered the alveolar saline. The animals challenged by alveolar instillation showed a greater permeability change (P less than 0.05). The bidirectional transfer of macromolecules indicates that alloxan produces a change in the permeability of the alveolar epithelium, allowing diffusional exchange of macromolecules. Since alveolar flooding in hemodynamic edema does not show a similar change in the permeability of the epithelial lining, alveolar flooding in alloxan edema is not due solely to an effect on the endothelial membrane, but also to a direct effect on the epithelial membrane.     

The functional role of rho and rho-associated coiled-coil forming protein kinase in eotaxin signaling of eosinophils

The CC chemokine eotaxin plays a pivotal role in local accumulation of eosinophils. Very little is known about the eotaxin signaling in eosinophils except the activation of the mitogen-activated protein (MAP) kinase family. The p21 G protein Rho and its substrate Rho-associated coiled-coil forming protein kinase (ROCK) regulate the formation of stress fibers and focal adhesions. In the present study, we studied the functional relevance of Rho and ROCK in eosinophils using the ROCK inhibitor (Y-27632) and exoenzyme C3, a specific Rho inhibitor. Eotaxin stimulates activation of Rho A and ROCK II in eosinophils. Exoenzyme C3 almost completely inhibited the ROCK activity, indicating that ROCK is downstream of Rho. We then examined the role of Rho and ROCK in eosinophil chemotaxis. The eotaxin-induced eosinophil chemotaxis was significantly inhibited by exoenzyme C3 or Y-27632. Because extracellular signal-regulated kinase (ERK)1/2 and p38 MAP kinases are activated by eotaxin and are critical for eosinophil chemotaxis, we investigated whether Rho and ROCK are upstream of these MAP kinases. C3 partially inhibited eotaxin-induced phosphorylation of ERK1/2 but not p38. In contrast, neither ERK1/2 nor p38 phosphorylation was abrogated by Y-27632. Both C3 and Y-27632 reduced reactive oxygen species production from eosinophils. We conclude that both Rho and ROCK are important for eosinophil chemotaxis and reactive oxygen species production. There is a dichotomy of downstream signaling pathways of Rho, namely, Rho-ROCK and Rho-ERK pathways. Taken together, eosinophil chemotaxis is regulated by multiple signaling pathways that involve at least ROCK, ERK, and p38 MAP kinase.     

Involvement of ROCK-mediated endothelial tension development in neutrophil-stimulated microvascular leakage

Neutrophil-induced coronary microvascular barrier dysfunction is an important pathophysiological event in heart disease. Currently, the precise cellular and molecular mechanisms of neutrophil-induced microvascular leakage are not clear. The aim of this study was to test the hypothesis that rho kinase (ROCK) increases coronary venular permeability in association with elevated endothelial tension. We assessed permeability to albumin (P(a)) in isolated porcine coronary venules and in coronary venular endothelial cell (CVEC) monolayers. Endothelial barrier function was also evaluated by measuring transendothelial electrical resistance (TER) of CVEC monolayers. In parallel, we measured isometric tension of CVECs grown on collagen gels. Transference of constitutively active (ca)-ROCK protein into isolated coronary venules or CVEC monolayers caused a significant increase in P(a) and decreased TER in CVECs. The ROCK inhibitor Y-27632 blocked the ca-ROCK-induced changes. C5a-activated neutrophils (10(6)/ml) also significantly elevated venular P(a), which was dose-dependently inhibited by Y-27632 and a structurally distinct ROCK inhibitor, H-1152. In CVEC monolayers, activated neutrophils increased permeability with a concomitant elevation in isometric tension, both of which were inhibited by Y-27632 or H-1152. Treatment with ca-ROCK also significantly increased CVEC monolayer permeability and isometric tension, coupled with actin polymerization and elevated phosphorylation of myosin regulatory light chain on Thr18/Ser19. The data suggest that during neutrophil activation, ROCK promotes microvascular leakage in association with actin-myosin-mediated tension development in endothelial cells.     

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.     

Rho kinase activation maintains high pulmonary vascular resistance in the ovine fetal lung

Mechanisms that maintain high pulmonary vascular resistance (PVR) in the fetal lung are poorly understood. Activation of the Rho kinase signal transduction pathway, which promotes actin-myosin interaction in vascular smooth muscle cells, is increased in the pulmonary circulation of adult animals with experimental pulmonary hypertension. However, the role of Rho kinase has not been studied in the fetal lung. We hypothesized that activation of Rho kinase contributes to elevated PVR in the fetus. To address this hypothesis, we studied the pulmonary hemodynamic effects of brief (10 min) intrapulmonary infusions of two specific Rho kinase inhibitors, Y-27632 (15-500 microg) and HA-1077 (500 microg), in chronically prepared late-gestation fetal lambs (n = 9). Y-27632 caused potent, dose-dependent pulmonary vasodilation, lowering PVR from 0.67 +/- 0.18 to 0.16 +/- 0.02 mmHg x ml(-1) x min(-1) (P < 0.01) at the highest dose tested without lowering systemic arterial pressure. Despite brief infusions, Y-27632-induced pulmonary vasodilation was sustained for 50 min. HA-1077 caused a similar fall in PVR, from 0.39 +/- 0.03 to 0.19 +/- 0.03 (P < 0.05). To study nitric oxide (NO)-Rho kinase interactions in the fetal lung, we tested the effect of Rho kinase inhibition on pulmonary vasoconstriction caused by inhibition of endogenous NO production with nitro-L-arginine (L-NA; 15-30 mg), a selective NO synthase antagonist. L-NA increased PVR by 127 +/- 73% above baseline under control conditions, but this vasoconstrictor response was completely prevented by treatment with Y-27632 (P < 0.05). We conclude that the Rho kinase signal transduction pathway maintains high PVR in the normal fetal lung and that activation of the Rho kinase pathway mediates pulmonary vasoconstriction after NO synthase inhibition. We speculate that Rho kinase plays an essential role in the normal fetal pulmonary circulation and that Rho kinase inhibitors may provide novel therapy for neonatal pulmonary hypertension.     

Transient rho-associated coiled-coil containing kinase (ROCK) inhibition on human retinal pigment epithelium results in persistent Rho/ROCK downregulation

Retinal pigment epithelium (RPE) cells is the outermost layer of the retina and RPE dysfunction is a key factor in the disease pathogenesis of age-related macular degeneration (AMD). Transplantation therapy using induced pluripotent stem cell (iPSC)-derived RPEs has recently received much attention as a treatment for AMD. Preserving these cells under the best possible conditions is important, and preservation methods using Y-27632 have been reported. Rho-associated coiled-coil containing kinase (ROCK) inhibitors are known to inhibit cell death, emerging as important drug candidates for stem cell differentiation and regenerative medicine. However, it has recently been shown that ROCK inhibitors may have a vasodilatory effect on human retinal arterioles, a side effect that should ideally be avoided in RPE transplantation. Although ROCK inhibitors hold great potential, optimizing efficacy while minimizing adverse reactions is critical for translation into a clinical treatment. We examined the effect of transient exposure of RPE cells to ROCK inhibitor Y-27632 to determine whether the extracellular presence of the drug is necessary for ongoing Rho/ROCK downregulation. Human RPE cells were subcultured as a suspension for 4 h in drug-free medium following exposure to Y-27632 for 2 h. A Y-27632 concentration of >10 μM improved cell survival beyond 4 h and cell proliferation in recovery culture medium. ROCK2 expression levels were specifically downregulated by Y-27632 in the Rho/ROCK signaling pathway. In conclusion, we demonstrated that the effect of Y-27632 is not dependent on its extracellular availability and can last beyond the 2 h of exposure. The lasting Rho/ROCK signaling pathway downregulation by Y-27632 suggests that RPE cell transplantation with ROCK inhibitor-free media is possible, which can minimize side effects to host tissue and have wider implications for transplantation methods requiring ROCK inhibition.     

Rho kinase activation plays a major role as a mediator of irreversible injury in reperfused myocardium

Intracellular signal transduction events in reperfusion following ischemia influence myocardial infarct development. Here we investigate the role of Rho kinase (ROCK) activation as a specific injury signal during reperfusion via attenuation of the reperfusion injury salvage kinase (RISK) pathway phosphatidylinositol 3-kinase (PI3K)/Akt/endothelial nitric oxide (NO) synthase (eNOS). Rat isolated hearts underwent 35 min of left coronary artery occlusion and 120 min of reperfusion. Phosphorylation of the ROCK substrate protein complex ezrin-radixin-moesin, assessed by immunoblotting and immunofluorescence, was used as a marker of ROCK activation. Infarct size was determined by tetrazolium staining, and terminal dUTP nick-end labeling (TUNEL) positivity was used as an index of apoptosis. The ROCK inhibitors fasudil or Y-27632 given 10 min before ischemia until 10 min after reperfusion reduced infarct size (control, 34.1 +/- 3.8%; 5 microM fasudil, 18.2 +/- 3.1%; 0.3 microM Y-27632, 19.4 +/- 4.4%; 5 microM Y-27632, 9.2 +/- 2.9%). When 5 microM Y-27632 was targeted specifically during early reperfusion, robust infarct limitation was observed (14.2 +/- 2.6% vs. control 33.4 +/- 4.4%, P<0.01). The protective action of Y-27632 given at reperfusion was attenuated by wortmannin (29.2 +/- 6.1%) and N(omega)-nitro-L-arginine methyl ester (30.4 +/- 5.7%), confirming a protective mechanism involving PI3K/Akt/NO. Ezrin-radixin-moesin phosphorylation in risk zone myocardium confirmed early and sustained ROCK activation during reperfusion and its inhibition by Y-27632. Inhibition of ROCK activation at reperfusion reduced the proportion of TUNEL-positive nuclei in the infarcted region. In conclusion, ROCK activation occurs specifically during early reperfusion. Inhibition of ROCK at reperfusion onset limits infarct size through an Akt/eNOS-dependent mechanism, suggesting that ROCK activation at reperfusion may be deleterious through suppression of the RISK pathway.     

The suppression of myosin light chain (MLC) phosphorylation during the response to lipopolysaccharide (LPS): beneficial or detrimental to endothelial barrier?

Sepsis-induced vascular leakage is a major underlying cause of the respiratory dysfunction seen in severe sepsis. Here, we studied the role of MLC phosphorylation in LPS-induced endothelial hyperpermeability and assessed how the changes in phospho-MLC distribution affect LPS-induced barrier dysfunction. We demonstrated that the changes in human lung microvascular endothelial permeability are preceded by the increase in intracellular calcium level, and increase in MYPT and MLC phosphorylation. Using the siRNA approach, we showed that both LPS-induced barrier dysfunction and MLC phosphorylation are attenuated by the depletion of the smooth muscle isoform of MLC kinase (MLCK) and Rho kinase 2 (ROCK2). Surprisingly, pharmacological inhibition of both ROCK1 and 2 with Y-27632 exacerbated LPS-induced drop in transendothelial resistance, although significantly decreasing MLC phosphorylation level. We next studied the involvement of protein kinase A (PKA)-dependent pathways in LPS-induced barrier dysfunction. We showed that LPS decreased the level of PKA-dependent phosphorylation in endothelial cells; and the pretreatment with forskolin or PKA activator bnz-cAMP counteracted this effect. Forskolin and bnz-cAMP also attenuated LPS-induced increase in MLC phosphorylation level. As we have shown earlier (Bogatcheva et al., 2009), forskolin and bnz-cAMP provide protection from LPS-induced barrier dysfunction. We compared the effects of bnz-cAMP and Y-27632 on phospho-MLC distribution and observed that while bnz-cAMP increased the association of the phospho-MLC signal with the cortical structures, Y-27632 decreased this association. These data indicate that an overall decrease in MLC phosphorylation could be either beneficial or detrimental to endothelial barrier, depending on the intracellular locale of major phospho-MLC changes.     

Involvement of RhoA and Rho kinase in neutrophil-stimulated endothelial hyperpermeability

Neutrophil-induced microvascular leakage is an early event in ischemic and inflammatory heart diseases. The specific signaling paradigm by which neutrophils increase microvascular permeability is not yet established. We investigated whether the small GTPase RhoA and its downstream effector Rho kinase mediate neutrophil-stimulated endothelial hyperpermeability. We assessed the effect of neutrophils on Rho activity in bovine coronary venular endothelial cells (CVEC) with a Rho-GTP pull-down assay. Permeability to FITC-albumin was evaluated using CVEC monolayers. We then tested the role of Rho kinase in the permeability response to neutrophils using two structurally distinct pharmacological inhibitors: Y-27632 and HA-1077. Furthermore, neutrophil-stimulated changes in endothelial F-actin organization were examined with fluorescence microscopy. The results show that C5a-activated neutrophils induced an increase in permeability coupled with RhoA activation in CVEC. Inhibition of Rho kinase with either Y-27632 or HA-1077 attenuated the hyperpermeability response. Rho kinase inhibition also attenuated increases in permeability stimulated by the neutrophil supernatant. In addition, activated neutrophils caused actin stress fiber formation in CVEC, which was diminished by either Y-27632 or HA-1077. These findings suggest that RhoA and Rho kinase are involved in the mediation of neutrophil-induced endothelial actin reorganization and barrier dysfunction.     

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.     

Glycine-extended gastrin stimulates cell proliferation and migration through a Rho- and ROCK-dependent pathway, not a Rac/Cdc42-dependent pathway

Both amidated gastrin (Gamide) and glycine-extended gastrin (Ggly) stimulate gastrointestinal cell proliferation and migration. Binding of Gamide to the cholecystokinin-2 receptor activates small GTP-binding proteins of the Rho family (Rho, Rac, and Cdc42), and dominant-negative mutants of Rho or Cdc42 block Gamide-stimulated cell proliferation and survival. In comparison, little is known about the Ggly signaling transduction pathway leading to cell proliferation and migration. The present study examined the roles of the small G proteins Rho, Rac, and Cdc42 in Ggly-induced proliferation and migration of the mouse gastric epithelial cell line IMGE-5. Ggly stimulated the activation of Rho and its downstream effector protein ROCK. The activation of Rho and ROCK mediated Ggly-induced cell proliferation and migration as inhibition of Rho by C3, or ROCK by Y-27632, completely blocked these effects of Ggly. Ggly also stimulated tyrosine phosphorylation of focal adhesion kinase, and stimulation was reversed by addition of C3 and Y-27632. In contrast to the effects of Rho and ROCK, inhibition of the Rac or Cdc42 pathways by expression of dominant-negative mutants of Rac or Cdc42 did not affect Ggly-induced cell proliferation and migration. These results demonstrate that Ggly stimulates IMGE-5 cell proliferation and migration through a Rho/ROCK-dependent pathway but not via Rac- or Cdc42-dependent pathways.     

Simultaneous measurement of fluid and protein permeability in isolated rabbit lungs during edema.

Fluid conductance and protein permeability have been studied in isolated perfused lung models of pulmonary edema. However, previous studies have not investigated changes of both fluid conductance and protein permeability in the same isolated lung preparation after injury. Arachidonic acid (AA) metabolites are involved in the inflammatory processes that lead to the development of pulmonary edema. The hemodynamic effects of AA have been well established; however, controversy exists concerning the ability of AA to alter the permeability of the pulmonary microvasculature to fluid and protein. The purpose of this study was to simultaneously determine whether transvascular fluid conductance and protein permeability are increased in isolated perfused rabbit lungs with pulmonary edema induced by AA. Indomethacin (80 microM) was added to the perfusate to inhibit the hemodynamic effects of AA and produce a pressure-independent model of pulmonary edema. Fluid conductance was assessed by determination of the capillary filtration coefficient (Kf), and protein permeability was evaluated by measurement of 125I-albumin clearance. The injection of AA (3 mg/200 ml of perfusate) into the pulmonary arterial catheter resulted in an increase in lung weight over the remaining 30-min experimental period. Kf (microliter.s-1 x cmH2O-1 x g dry lung-1) was increased (P < 0.05) in AA-treated lungs at 10 and 30 min post-AA injection when compared with control lungs and baseline values (determined 10 min before AA injection). Albumin clearance was also greater (P < 0.05) in lungs that received AA. 125I-albumin clearance was measured at different rates of fluid flux produced by elevation of venous pressure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Attenuation of acute hypoxic pulmonary vasoconstriction and hypoxic pulmonary hypertension in mice by inhibition of Rho-kinase

RhoA GTPase mediates a variety of cellular responses, including activation of the contractile apparatus, growth, and gene expression. Acute hypoxia activates RhoA and, in turn, its downstream effector, Rho-kinase, and previous studies in rats have suggested a role for Rho/Rho-kinase signaling in both acute and chronically hypoxic pulmonary vasoconstriction. We therefore hypothesized that activation of Rho/Rho-kinase in the pulmonary circulation of mice contributes to acute hypoxic pulmonary vasoconstriction and chronic hypoxia-induced pulmonary hypertension and vascular remodeling. In isolated, salt solution-perfused mouse lungs, acute administration of the Rho-kinase inhibitor Y-27632 (1 x 10(-5) M) attenuated hypoxic vasoconstriction as well as that due to angiotensin II and KCl. Chronic treatment with Y-27632 (30 mg x kg(-1) x day(-1)) via subcutaneous osmotic pump decreased right ventricular systolic pressure, right ventricular hypertrophy, and neomuscularization of the distal pulmonary vasculature in mice exposed to hypobaric hypoxia for 14 days. Analysis of a small number of proximal pulmonary arteries suggested that Y-27632 treatment reduced the level of phospho-CPI-17, a Rho-kinase target, in hypoxic lungs. We also found that endothelial nitric oxide synthase protein in hypoxic lungs was augmented by Y-27632, suggesting that enhanced nitric oxide production might have played a role in the Y-27632-induced attenuation of chronically hypoxic pulmonary hypertension. In conclusion, Rho/Rho-kinase activation is important in the effects of both acute and chronic hypoxia on the pulmonary circulation of mice, possibly by contributing to both vasoconstriction and vascular remodeling.