Synthetic Amphipathic Helical Peptides Targeting CD36 Attenuate Lipopolysaccharide-Induced Inflammation and Acute Lung Injury.

Synthetic amphipathic helical peptides (SAHPs) designed as apolipoprotein A-I mimetics are known to bind to class B scavenger receptors (SR-Bs), SR-BI, SR-BII, and CD36, receptors that mediate lipid transport and facilitate pathogen recognition. In this study, we evaluated SAHPs, selected for targeting human CD36, by their ability to attenuate LPS-induced inflammation, endothelial barrier dysfunction, and acute lung injury (ALI). L37pA, which targets CD36 and SR-BI equally, inhibited LPS-induced IL-8 secretion and barrier dysfunction in cultured endothelial cells while reducing lung neutrophil infiltration by 40% in a mouse model of LPS-induced ALI. A panel of 20 SAHPs was tested in HEK293 cell lines stably transfected with various SR-Bs to identify SAHPs with preferential selectivity toward CD36. Among several SAHPs targeting both SR-BI/BII and CD36 receptors, ELK-B acted predominantly through CD36. Compared with L37pA, 5A, and ELK SAHPs, ELK-B was most effective in reducing the pulmonary barrier dysfunction, neutrophil migration into the lung, and lung inflammation induced by LPS. We conclude that SAHPs with relative selectivity toward CD36 are more potent at inhibiting acute pulmonary inflammation and dysfunction. These data indicate that therapeutic strategies using SAHPs targeting CD36, but not necessarily mimicking all apolipoprotein A-I functions, may be considered a possible new treatment approach for inflammation-induced ALI and pulmonary edema.

Control of vascular permeability by atrial natriuretic peptide via a GEF-H1-dependent mechanism.

Microtubule (MT) dynamics is involved in a variety of cell functions, including control of the endothelial cell (EC) barrier. Release of Rho-specific nucleotide exchange factor GEF-H1 from microtubules activates the Rho pathway of EC permeability. In turn, pathologic vascular leak can be prevented by treatment with atrial natriuretic peptide (ANP). This study investigated a novel mechanism of vascular barrier protection by ANP via modulation of GEF-H1 function. In pulmonary ECs, ANP suppressed thrombin-induced disassembly of peripheral MT and attenuated Rho signaling and cell retraction. ANP effects were mediated by the Rac1 GTPase effector PAK1. Activation of Rac1-PAK1 promoted PAK1 interaction with the Rho activator GEF-H1, inducing phosphorylation of total and MT-bound GEF-H1 and leading to attenuation of Rho-dependent actin remodeling. In vivo, ANP attenuated lung injury caused by excessive mechanical ventilation and TRAP peptide (TRAP/HTV), which was further exacerbated in ANP(-/-) mice. The protective effects of ANP against TRAP/HTV-induced lung injury were linked to the increased pool of stabilized MT and inactivation of Rho signaling via ANP-induced, PAK1-dependent inhibitory phosphorylation of GEF-H1. This study demonstrates a novel protective mechanism of ANP against pathologic hyperpermeability and suggests a novel pharmacological intervention for the prevention of increased vascular leak via PAK1-dependent modulation of GEF-H1 activity.

Iloprost improves endothelial barrier function in lipopolysaccharide-induced lung injury.

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.

Novel role of stathmin in microtubule-dependent control of endothelial permeability.

Microtubule (MT) dynamics in vascular endothelium are modulated by vasoactive mediators and are critically involved in the control of endothelial cell (EC) permeability via Rho GTPase-dependent crosstalk with the actin cytoskeleton. However, the role of regulators in MT stability in these mechanisms remains unclear. This study investigated the involvement of the MT-associated protein stathmin in the mediation of agonist-induced permeability in EC cultures and vascular leak in vivo. Thrombin treatment of human pulmonary ECs induced rapid dephosphorylation and activation of stathmin. Inhibition of stathmin activity by small interfering RNA-based knockdown or cAMP-mediated phosphorylation abrogated thrombin-induced F-actin remodeling and Rho-dependent EC hyperpermeability, while expression of a phosphorylation-deficient stathmin mutant exacerbated thrombin-induced EC barrier disruption. Stathmin suppression preserved the MT network against thrombin-induced MT disassembly and release of Rho-specific guanine nucleotide exchange factor, GEF-H1. The protective effects of stathmin knockdown were observed in vivo in the mouse 2-hit model of ventilator-induced lung injury and were linked to MT stabilization and down-regulation of Rho signaling in the lung. These results demonstrate the mechanism of stathmin-dependent control of MT dynamics, Rho signaling, and permeability and suggest novel potential pharmacological interventions in the prevention of increased vascular leak via modulation of stathmin activity.

Oxidative stress contributes to lung injury and barrier dysfunction via microtubule destabilization.

Oxidative stress is an important part of host innate immune response to foreign pathogens, such as bacterial LPS, but excessive activation of redox signaling may lead to pathologic endothelial cell (EC) activation and barrier dysfunction. Microtubules (MTs) play an important role in agonist-induced regulation of vascular endothelial permeability, but their impact in modulation of inflammation and EC barrier has not been yet investigated. This study examined the effects of LPS-induced oxidative stress on MT dynamics and the involvement of MTs in the LPS-induced mechanisms of Rho activation, EC permeability, and lung injury. LPS treatment of pulmonary vascular EC induced elevation of reactive oxygen species (ROS) and caused oxidative stress associated with EC hyperpermeability, cytoskeletal remodeling, and formation of paracellular gaps, as well as activation of Rho, p38 stress kinase, and NF-κB signaling, the hallmarks of endothelial barrier dysfunction. LPS also triggered ROS-dependent disassembly of the MT network, leading to activation of MT-dependent signaling. Stabilization of MTs with epothilone B, or inhibition of MT-associated guanine nucleotide exchange factor (GEF)-H1 activity by silencing RNA-mediated knockdown, suppressed LPS-induced EC barrier dysfunction in vitro, and attenuated vascular leak and lung inflammation in vivo. LPS disruptive effects were linked to activation of Rho signaling caused by LPS-induced MT disassembly and release of Rho-specific GEF-H1 from MTs. These studies demonstrate, for the first time, the mechanism of ROS-induced Rho activation via destabilization of MTs and GEF-H1-dependent activation of Rho signaling, leading to pulmonary EC barrier dysfunction and exacerbation of LPS-induced inflammation.

Differential regulation of endothelial cell permeability by high and low doses of oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine.

The generation of phospholipid oxidation products in atherosclerosis, sepsis, and lung pathologies affects endothelial barrier function, which exerts significant consequences on disease outcomes in general. Our group previously showed that oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (OxPAPC) at low concentrations increases endothelial cell (EC) barrier function, but decreases it at higher concentrations. In this study, we determined the mechanisms responsible for the pulmonary endothelial cell barrier dysfunction induced by high OxPAPC concentrations. OxPAPC at a range of 5-20 µg/ml enhanced EC barriers, as indicated by increased transendothelial electrical resistance. In contrast, higher OxPAPC concentrations (50-100 µg/ml) rapidly increased EC permeability, which was accompanied by increased total cell protein tyrosine (Tyr) phosphorylation, phosphorylation at Tyr-418, the activation of Src kinase, and the phosphorylation of adherens junction (AJ) protein vascular endothelial cadherin (VE-cadherin) at Tyr-731 and Tyr-658, which was not observed in ECs stimulated with low OxPAPC doses. The early tyrosine phosphorylation of VE-cadherin was linked to the dissociation of VE-cadherin-p120-catenin/ß-catenin complexes and VE-cadherin internalization, whereas low OxPAPC doses promoted the formation of VE-cadherin-p120-catenin/ß-catenin complexes. High but not low doses of OxPAPC increased the production of reactive oxygen species (ROS) and protein oxidation. The inhibition of Src by PP2 and ROS production by N-acetyl cysteine inhibited the disassembly of VE-cadherin-p120-catenin complexes, and attenuated high OxPAPC-induced EC barrier disruption. These results show the differential effects of OxPAPC doses on VE-cadherin-p120-catenin complex assembly and EC barrier function. These data suggest that the rapid tyrosine phosphorylation of VE-cadherin and other potential targets mediated by Src and ROS-dependent mechanisms plays a key role in the dissociation of AJ complexes and EC barrier dysfunction induced by high OxPAPC doses.

Afadin controls p120-catenin-ZO-1 interactions leading to endothelial barrier enhancement by oxidized phospholipids.

Afadin is a novel regulator of epithelial cell junctions assembly. However, its role in the formation of endothelial cell junctions and the regulation of vascular permeability remains obscure. We previously described protective effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) in the in vitro and in vivo models of lung endothelial barrier dysfunction and acute lung injury, which were mediated by Rac GTPase. This study examined a role of afadin in the OxPAPC-induced enhancement of interactions between adherens junctions and tight junctions as a novel mechanism of endothelial cell (EC) barrier preservation. OxPAPC induced Rap1-dependent afadin accumulation at the cell periphery and Rap1-dependent afadin interaction with adherens junction and tight junction proteins p120-catenin and ZO-1, respectively. Afadin knockdown using siRNA or ectopic expression of afadin mutant lacking Rap1 GTPase binding domain suppressed OxPAPC-induced EC barrier enhancement and abolished barrier protective effects of OxPAPC against thrombin-induced EC permeability. Afadin knockdown also abolished protective effects of OxPAPC against ventilator-induced lung injury in vivo. These results demonstrate for the first time a critical role of afadin in the regulation of vascular barrier function in vitro and in vivo via coordination of adherens junction-tight junction interactions.

Stimulation of Rho signaling by pathologic mechanical stretch is a "second hit" to Rho-independent lung injury induced by IL-6.

Most patients with acute lung injury (ALI) and acute respiratory distress syndrome of septic and nonseptic nature require assisted ventilation with positive pressure, which at suboptimal range may further exacerbate lung dysfunction. Previous studies described enhancement of agonist-induced Rho GTPase signaling and endothelial cell (EC) permeability in EC cultures exposed to pathologically relevant cyclic stretch (CS) magnitudes. This study examined a role of pathologic CS in modulation of pulmonary EC permeability caused by IL-6, a cytokine increased in sepsis and acting in a Rho-independent manner. IL-6 increased EC permeability, which was associated with activation of Jak/signal transducers and activators of transcription, p38 MAP kinase, and NF-κB signaling and was augmented by EC exposure to 18% CS. Rho kinase inhibitor Y-27632 suppressed the synergistic effect of 18% CS on IL-6-induced EC monolayer disruption but did not alter the IL-6 effects on static EC culture. 18% CS also increased IL-6-induced ICAM-1 expression by pulmonary EC and neutrophil adhesion, which was attenuated by Y-27632. Intratracheal IL-6 administration in C57BL/6J mice increased protein content and cell count in bronchoalveolar lavage fluid. These changes were augmented by high tidal volume mechanical ventilation (HTV; 30 ml/kg, 4 h). Intravenous injection of Y-27632 suppressed IL6/HTV-induced lung injury. In conclusion, this study proposes a novel mechanism contributing to two-hit model of ALI: in addition to synergistic effects on Rho-dependent endothelial hyper-permeability triggered by thrombin, TNFα, LPS, or other agonists, ventilator-induced lung injury-relevant CS may also exacerbate Rho-independent mechanisms of EC permeability induced by other inflammatory mediators such as IL-6 via mechanisms involving Rho activity.

p190RhoGAP mediates protective effects of oxidized phospholipids in the models of ventilator-induced lung injury.

Products resulting from oxidation of cell membrane phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (OxPAPC) exhibit potent protective effects against lung endothelial cell (EC) barrier dysfunction caused by pathologically relevant mechanical forces and inflammatory agents. These effects were linked to enhancement of peripheral cytoskeleton and cell adhesion interactions mediated by small GTPase Rac and inhibition of Rho-mediated barrier-disruptive signaling. However, the mechanism of OxPAPC-induced, Rac-dependent Rho downregulation critical for vascular barrier protection remains unclear. This study tested the hypothesis that Rho negative regulator p190RhoGAP is essential for OxPAPC-induced lung barrier protection against ventilator-induced lung injury (VILI), and investigated potential mechanism of p190RhoGAP targeting to adherens junctions (AJ) via p120-catenin. OxPAPC induced peripheral translocation of p190RhoGAP, which was abolished by knockdown of Rac-specific guanine nucleotide exchange factors Tiam1 and Vav2. OxPAPC also induced Rac-dependent tyrosine phosphorylation and association of p190RhoGAP with AJ protein p120-catenin. siRNA-induced knockdown of p190RhoGAP attenuated protective effects of OxPAPC against EC barrier compromise induced by thrombin and pathologically relevant cyclic stretch (18% CS). In vivo, p190RhoGAP knockdown significantly attenuated protective effects of OxPAPC against ventilator-induced lung vascular leak, as detected by increased cell count and protein content in the bronchoalveolar lavage fluid, and tissue neutrophil accumulation in the lung. These results demonstrate for the first time a key role of p190RhoGAP for the vascular endothelial barrier protection in VILI.

Knockdown of FABP5 mRNA decreases cellular cholesterol levels and results in decreased apoB100 secretion and triglyceride accumulation in ARPE-19 cells.

To maintain normal retinal function, retinal pigment epithelial (RPE) cells engulf photoreceptor outer segments (ROS) enriched in free fatty acids (FFAs). We have previously demonstrated fatty acid-binding protein 5 (FABP5) downregulation in the RPE/choroidal complex in a mouse model of aging and early age-related macular degeneration. FABPs are involved in intracellular transport of FFAs and their targeting to specific metabolic pathways. To elucidate the role of FABP5 in lipid metabolism, the production of the FABP5 protein in a human RPE cell line was inhibited using RNA interference technology. As a result, the levels of cholesterol and cholesterol ester were decreased by about 40%, whereas FFAs and triglycerides were increased by 18 and 67% after siRNA treatment, respectively. Some species of phospholipids were decreased in siRNA-treated cells. Cellular lipid droplets were evident and apoB secretion was decreased by 76% in these cells. Additionally, we discovered that ARPE-19 cells could synthesize and secrete Apolipoprotein B100 (apoB100), which may serve as a backbone structure for the formation of lipoprotein particles in these cells. Our results indicate that FABP5 mRNA knockdown results in the accumulation of cellular triglycerides, decreased cholesterol levels, and reduced secretion of apoB100 protein and lipoprotein-like particles. These observations indicated that FABP5 plays a critical role in lipid metabolism in RPE cells, suggesting that FABP5 downregulation in the RPE/choroid complex in vivo might contribute to aging and early age-related macular degeneration.

Apolipoprotein B100 secretion by cultured ARPE-19 cells is modulated by alteration of cholesterol levels.

Cholesteryl ester rich apolipoprotein B100 (apoB100) lipoproteins accumulate in Bruch's membrane before the development of age-related macular degeneration. It is not known if these lipoproteins come from the circulation or local ocular tissue. Emerging, but incomplete evidence suggests that the retinal pigmented epithelium (RPE) can secrete lipoproteins. The purpose of this investigation was to determine (i) whether human RPE cells synthesize and secrete apoB100, and (ii) whether this secretion is driven by cellular cholesterol, and if so, (iii) whether statins inhibit this response. The established, human derived ARPE-19 cells challenged with 0-0.8 mM oleic acid accumulated cellular cholesterol, but not triglycerides. Oleic acid increased the amount of apoB100 protein recovered from the medium by both western blot analysis and (35) S-radiolabeled immunoprecipitation while negative stain electron microscopy showed lipoprotein-like particles. Of nine statins evaluated, lipophilic statins induced HMG-CoA reductase mRNA expression the most. The lipophilic Cerivastatin (5 µM) reduced cellular cholesterol by 39% and abrogated apoB100 secretion by 3-fold. In contrast, the hydrophilic statin Pravastatin had minimal effect on apoB100 secretion. These data suggest that ARPE-19 cells synthesize and secrete apoB100 lipoproteins, that this secretion is driven by cellular cholesterol, and that statins can inhibit apoB100 secretion by reducing cellular cholesterol.

Oxidized low density lipoproteins induce a pathologic response by retinal pigmented epithelial cells.

The accumulation of apolipoprotein B100 lipoproteins in Bruch membrane is an early event thought to promote age-related macular degeneration (AMD). Immunohistochemistry using an anti-oxidized low density lipoprotein antibody on 10 AMD specimens showed staining in Bruch membrane including basal deposits, a marker of AMD. To determine whether retinal pigmented epithelial cells develop a pathologic phenotype after interaction with lipoproteins, ARPE-19 cells were exposed to low density lipoproteins (LDL) or oxidized LDLs (oxLDL). Analysis using the Affymetrix U133 Plus 2.0 (Affymetrix, Inc., Santa Clara, CA, USA) gene chip showed physiological and pathological transcriptional responses after LDL and oxLDL treatment, respectively. LDL induced a down-regulation of cholesterol biosynthesis genes while oxLDL induced transcriptional alterations in genes related to lipid metabolism, oxidative stress, inflammation and apoptosis. Electrospray mass spectrometry showed that oxLDL, but not LDL induced large cellular increases of sphingomyelin, ceramides, and cholesteryl esters. With TUNEL labeling, oxLDL caused 14.6% apoptosis compared to <1% after LDL. Addition of an inhibitor of sphingomyelin synthase inhibited this apoptosis by 41%. These data support the hypothesis that oxidized lipoproteins are one trigger for initiating early events in the pathogenesis of AMD.

Release of pyridoxal 5'-phosphate upon unfolding of mitochondrial aspartate aminotransferase.

Dimeric mitochondrial aspartate aminotransferase (mAAT) contains a molecule of pyridoxal 5'-phosphate (PLP) tightly attached to each of its two identical active sites. The presence of this natural reporter allows us to study separately local perturbations in the architecture of this critical region of the molecule during unfolding. Upon unfolding of the enzyme with guanidine hydrochloride (GdnHCl), the coenzyme is completely released from the active site. The transition midpoint for the dissociation of PLP is 1.4+/-0.02 M when determined by size-exclusion chromatography (SEC) and 1.6+/-0.02 M when the protein-bound PLP is estimated by electrospray mass spectrometry (ESI-MS). In both cases the transition midpoint is higher than that of inactivation (1.3+/-0.01 M). On the other hand, the midpoint of the unfolding transition obtained by monitoring changes in ellipticity at 356 nm, which reflects the asymmetric environment of the PLP cofactor at the active site, is 1.19+/-0.011 M guanidine. These results indicate that the unfolding of mAAT is a multi-step process which includes an intermediate containing bound PLP but lacking catalytic activity.

Light-induced oxidative stress in choroidal endothelial cells in mice.

PURPOSE: Although light-induced oxidative stress in the retina has been extensively reported, little information regarding light-induced oxidative stress in choroidal endothelial cells (CECs) is available. In the current study, light-induced DNA oxidation and the activation of nuclear factor-kappaB (NF-kappaB), a major oxidative responsive transcription factor, were investigated in mouse CECs. METHODS: Mice were exposed to green light. Light-induced DNA oxidation in CECs was detected by in situ 8-hydroxy-2-deoxyguanosine (8-oxo-dG) immunolabeling. CECs were isolated from the retinal pigment epithelium (RPE)/choroid by using immunomagnetic beads. The isolated CECs were immunochemically characterized by the expression of endothelial markers, CD31, and P1H12. The quality of total RNA from CECs was assessed by a bioanalyzer and RT-PCR. NF-kappaB activation in situ and in isolated CECs was investigated. RESULTS: After a 3-hour exposure to light, the immunoreactivity to anti-8-oxo-dG antibody or anti-NF-kappaB p65 antibody in CECs in situ was significantly increased when compared with unexposed mice. Isolated CECs expressed CD31 and P1H12. The 28S/18S rRNA ratio of RNA isolated from CECs was 1.5:1. CD31 and von Willebrand Factor (vWF) transcripts were predominantly expressed in the RNA from isolated CECs. IkappaBalpha was more heavily phosphorylated in light-exposed than untreated CECs. IkappaBalpha expression levels were increased fivefold in isolated CECs after exposure to light compared to unexposed control subjects. CONCLUSIONS: Exposure to light induces oxidative stress in CECs in vivo. A method for CEC isolation from the mouse RPE/choroid with preservation of RNA quality has been developed. The results of this study may facilitate the ability to identify CEC-specific genes and gene products that respond to photo-oxidative stress.

Retinal abnormalities associated with the G90D mutation in opsin.

Several mutations in the opsin gene have been associated with congenital stationary night blindness, considered to be a relatively nonprogressive disorder. In the present study, we examined the structural and functional changes induced by one of these mutations, i.e., substitution of aspartic acid for glycine at position 90 (G90D). Transgenic mice were created in which the ratio of transgenic opsin transcript to endogenous was 0.5:1, 1.7:1, or 2.5:1 and were studied via light and electron microscopy, immunocytochemistry, electroretinography (ERG), and spectrophotometry. Retinas with transgenic opsin levels equivalent to one endogenous allele (G0.5) appeared normal for a period of about 3-4 months, but at later ages there were disorganized, shortened rod outer segments (ROS), and a loss of photoreceptor nuclei. Higher levels of G90D opsin expression produced earlier signs of retinal degeneration and more severe disruption of photoreceptor morphology. Despite these adverse effects, the mutation had a positive effect on the retinas of rhodopsin knockout (R-/-) mice, whose visual cells fail to form ROS and rapidly degenerate. Incorporation of the transgene in the null background (G+/-/R-/- or G+/+/R-/-) led to the development of ROS containing G90D opsin and prolonged survival of photoreceptors. Absorbance spectra measured both in vitro and in situ showed a significant reduction of more than 90% in the amount of light-sensitive pigment in the retinas of G+/+/R-/- mice, and ERG recordings revealed a >1 log unit loss in sensitivity. However, the histological appearances of the retinas of these mice show no significant loss of photoreceptors and little change in the lengths of their outer segments. These findings suggest that much of the ERG sensitivity loss derives from the reduced quantal absorption that results from a failure of G90D opsin to bind to its chromophore and form a normal complement of light-sensitive visual pigment.

NF-kappaB activation in light-induced retinal degeneration in a mouse model.

PURPOSE: To investigate the modulation of nuclear factor (NF)-kappaB in light-induced photoreceptor degeneration in a mouse model. METHODS: Mice were exposed to intense green light. Light-induced activation of NF-kappaB and its nuclear localization were studied by immunohistochemistry. The NF-kappaB DNA-binding activity in the retinas after exposure to light was measured by electrophoretic mobility shift assay (EMSA). Nuclear transactivation of NF-kappaB in the photoreceptor cells was determined by quantitative real-time (qRT)-PCR. The amount of NF-kappaB p65 in the photoreceptor cells after exposure to light was assessed by Western blot analysis. To obtain more photoreceptor-specific information, microdissected photoreceptor cells were used in some studies. RESULTS: By an immunohistochemical method, the perinuclear region of the photoreceptor cells was heavily labeled with an antibody to activated NF-kappaB after a 1-hour exposure to light. Nuclear localization of NF-kappaB in the photoreceptor nucleus was seen at 12 hours. In the experiments involving 3 hours of exposure to light followed by recovery in the dark, nuclear localization of NF-kappaB was also noted after 12 hours' recovery in the dark. During continuous exposure to light, the NF-kappaB DNA-binding activity gradually increased and reached its maximum at 12 hours. There was an increase of NF-kappaB p65 protein at 3 hours. The mRNA levels of IkappaBalpha were upregulated after 6 hours' exposure to light. CONCLUSIONS: Intense light activated NF-kappaB in the photoreceptor cells in vivo, increased the NF-kappaB DNA-binding activity, and increased the expression of mRNA of IkappaBalpha, a target gene of NF-kappaB.

Light-induced photoreceptor degeneration may involve the NF kappa B/caspase-1 pathway in vivo.

PURPOSE: To determine the role of nuclear factor-kappaB (NFkappaB) in light-induced photoreceptor degeneration. METHODS: Dark-adapted BALB/cJ mice, 4-8 weeks, were exposed to an intense green light (3.1-3.5 klux) for 1, 3, 6, 9, 12, or 24 h and killed immediately after exposure. The photoreceptor apoptosis was detected by TUNEL. Co-localization of NFkappaB p65 immunoreactivity and TUNEL in photoreceptor cells was detected by double immunolabeling. The protein levels of X-linked inhibitor of apoptosis protein (XIAP), Bcl-xL, caspase-1, and opsin after light exposure were analyzed by Western blot analysis. In addition, the initiation of NFkappaB activation was assessed by measuring the increase in phosphorylated IkappaBalpha (pIkappaBalpha). Immunohistochemical localization of caspase-1 was also performed on the mouse retinas. RESULTS: Co-localization of NFkappaB p65 immunoreactivity with TUNEL was observed in scattered photoreceptor cells after 24 h of light exposure. The amount of pIkappaBalpha was increased after 1 h of light exposure, and in parallel, the amounts of XIAP and Bcl-xL were increased at 1 h. In contrast, caspase-1 did not increase until after 6 h of light exposure. Caspase-1-immunolabeling was observed in scattered photoreceptor cells after 3 h of light exposure but was markedly increased in many more cells at 6 h. CONCLUSIONS: These findings suggest that NFkappaB may play an anti-apoptotic role in the early response to light stress and that photoreceptor apoptosis induced by light stress may be mediated through an NFkappaB/caspase-1 pathway.

[Activation of NF-kappa B in the mouse retina after ischemia reperfusion].

OBJECTIVE: To investigate the expression of NF-kappa B following retinal ischemia and reperfusion injury in mice. METHODS: Retinal ischemia was induced by elevation of intraocular pressure. Retinal degeneration and atrophy were quantified by an image analysis system. Immunohistochemistry using p65 monoclonal antibody was performed on the retina and co-related with TUNEL labeling. RESULTS: Inner retinal thickness was increased in the initial 24 hours following retinal ischemia and was ascribed to tissue edema, but was significantly decreased by 168 hours after reperfusion. Six hours after retinal ischemia, p65 immunoreactivity was increased in the ganglion cell and the inner nuclear layers, reached a peak at 24 hours, and was parallel to TUNEL labeling. Double labeling with p65 and TUNEL showed partial co-localization of p65 and TUNEL labeling, predominantly in the inner nuclear layer. CONCLUSIONS: Activation of NF-kappa B appears to play an important role in retinal degeneration following retinal ischemia and reperfusion injury. The pro- and anti-apoptotic effects of NF-kappa B after retinal ischemia are being further investigated.

Stiffness-activated GEF-H1 expression exacerbates LPS-induced lung inflammation.

Acute lung injury (ALI) is accompanied by decreased lung compliance. However, a role of tissue mechanics in modulation of inflammation remains unclear. We hypothesized that bacterial lipopolysacharide (LPS) stimulates extracellular matrix (ECM) production and vascular stiffening leading to stiffness-dependent exacerbation of endothelial cell (EC) inflammatory activation and lung barrier dysfunction. Expression of GEF-H1, ICAM-1, VCAM-1, ECM proteins fibronectin and collagen, lysyl oxidase (LOX) activity, interleukin-8 and activation of Rho signaling were analyzed in lung samples and pulmonary EC grown on soft (1.5 or 2.8 kPa) and stiff (40 kPa) substrates. LPS induced EC inflammatory activation accompanied by expression of ECM proteins, increase in LOX activity, and activation of Rho signaling. These effects were augmented in EC grown on stiff substrate. Stiffness-dependent enhancement of inflammation was associated with increased expression of Rho activator, GEF-H1. Inhibition of ECM crosslinking and stiffening by LOX suppression reduced EC inflammatory activation and GEF-H1 expression in response to LPS. In vivo, LOX inhibition attenuated LPS-induced expression of GEF-H1 and lung dysfunction. These findings present a novel mechanism of stiffness-dependent exacerbation of vascular inflammation and escalation of ALI via stimulation of GEF-H1-Rho pathway. This pathway represents a fundamental mechanism of positive feedback regulation of inflammation.