Potential role of dietary flavonoids in reducing microvascular endothelium vulnerability to oxidative and inflammatory insults ( small star,filled)

Although antioxidant systems help control the level of reactive oxygen species they may be overwhelmed during periods of oxidative stress. Evidence suggests that oxidative stress components as well as inflammatory mediators may be involved in the pathogenesis of vascular disorders, where localized markers of oxidative damage have been found. In this regard we investigated the putative antioxidant and anti-inflammatory effects of blueberry and cranberry anthocyanins and hydroxycinnamic acids against H(2)O(2) and TNFalpha induced damage to human microvascular endothelial cells. Polyphenols from both berries were able to localize into endothelial cells subsequently reducing endothelial cells vulnerability to increased oxidative stress at both the membrane and cytosol level. Furthermore, berry polyphenols also reduced TNFalpha induced up-regulation of various inflammatory mediators (IL-8, MCP-1 and ICAM-1) involved in the recruitment of leukocytes to sites of damage or inflammation along the endothelium. In conclusion, polyphenols isolated from both blueberry and cranberry were able to afford protection to endothelial cells against stressor induced up-regulation of oxidative and inflammatory insults. This may have beneficial actions against the initiation and development of vascular diseases and be a contributing factor in the reduction of age-related deficits in neurological impairments previously reported by us.     

Structural requirements of anthocyanins in relation to inhibition of endothelial injury induced by oxidized low-density lipoprotein and correlation with radical scavenging activity

Anthocyanins may play an important role in atherosclerosis prevention. However, the structure-function relationships are not well understood. The objective of this study was to compare the inhibitory effect of 21 anthocyanins against oxidized low-density lipoprotein-induced endothelial injury to understand the relationship between anthocyanin chemical structure and the endothelial protective properties, measured as cell viability, MDA production and NO release. Additionally, the intracellular anti-radical activity of the selected anthocyanins was investigated to identify the correlation with endothelial protection. Our results provide evidence that the number of -OH in total or in B-ring, 3′,4′-ortho-dihydroxyl and 3-hydroxyl are the main structural requirements of anthocyanins in suppressing oxidative stress-induced endothelial injury and such inhibitory effect was significantly correlated with the intracellular radical scavenging activity.     

Resveratrol increases vascular oxidative stress resistance

Epidemiological studies suggest that Mediterranean diets rich in resveratrol are associated with reduced risk of coronary artery disease. However, the mechanisms by which resveratrol exerts its vasculoprotective effects are not completely understood. Because oxidative stress and endothelial cell injury play a critical role in vascular aging and atherogenesis, we evaluated whether resveratrol inhibits oxidative stress-induced endothelial apoptosis. We found that oxidized LDL and TNF-alpha elicited significant increases in caspase-3/7 activity in endothelial cells and cultured rat aortas, which were prevented by resveratrol pretreatment (10(-6)-10(-4) mol/l). The protective effect of resveratrol was attenuated by inhibition of glutathione peroxidase and heme oxygenase-1, suggesting a role for antioxidant systems in the antiapoptotic action of resveratrol. Indeed, resveratrol treatment protected cultured aortic segments and/or endothelial cells against increases in intracellular H(2)O(2) levels and H(2)O(2)-mediated apoptotic cell death induced by oxidative stressors (exogenous H(2)O(2), paraquat, and UV light). Resveratrol treatment also attenuated UV-induced DNA damage (comet assay). Resveratrol treatment upregulated the expression of glutathione peroxidase, catalase, and heme oxygenase-1 in cultured arteries, whereas it had no significant effect on the expression of SOD isoforms. Resveratrol also effectively scavenged H(2)O(2) in vitro. Thus resveratrol seems to increase vascular oxidative stress resistance by scavenging H(2)O(2) and preventing oxidative stress-induced endothelial cell death. We propose that the antioxidant and antiapoptotic effects of resveratrol, together with its previously described anti-inflammatory actions, are responsible, at least in part, for its cardioprotective effects.     

Protective effects of ellagic and chlorogenic acids against oxidative stress in PC12 cells

Following exposure of differentiated neuronal PC12 cells to either t-BHP, hydrogen peroxide (H₂O₂) or FeSO₄ various kinds of reactive oxygen species (ROS) are generated leading to oxidative injury. The protective effects of two plant polyphenols, ellagic (EC) and chlorogenic acid (CGA), as well as of two metabolites, caffeic acid (CA) and ferulic acid (FA), were investigated in preincubation and coincubation experiments with respect to the following parameters: prevention of cell death, GSH depletion, lipid peroxidation and ROS formation.

The polyphenols more efficiently suppressed cytotoxicity and loss of GSH caused by peroxides than by iron, particularly in preincubation. Lipid peroxidation which increased much stronger in response to FeSO₄ was counteracted completely by the polyphenols. In case of iron, however, only coincubation was effective. EA and CGA and the metabolites CA and FA showed excellent elimination of ROS induced by all stressors. These findings suggest that two dietary antioxidants, EA and CGA, may have protective properties against oxidative stress induced in CNS.     

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.     

Resveratrol attenuates oxLDL-stimulated NADPH oxidase activity and protects endothelial cells from oxidative functional damages.

trans-Resveratrol (RSV) has been shown to have cardioprotective effect during ischemia-reperfusion through reactive oxygen species (ROS)-scavenging activity. Elevated ROS has been implicated in the initiation and progression of atherosclerosis. The nicotinamide adenine dinucleotide phosphate oxidase (NOX) is a major source of vascular ROS formation. In the present study, we show that exposure of vascular endothelial cells (EC) to oxidized low-density lipoproteins (oxLDL) results in elevations of NOX activity and cellular ROS levels. The oxLDL effects are effectively suppressed by RSV or astringinin (AST), either before or after oxLDL exposure. In this study, we show that RSV or AST treatment appears to suppress NOX activity by reducing the membrane association of gp91(phox) and Rac1, two protein species required for the assembly of active NOX complex. Exposure to RSV or AST protects EC from oxidative functional damages, including antiplatelet activity and mononucleocyte adhesion. In addition, ANG II-induced NOX activation is also attenuated. These results suggest that RSV or AST protects EC from oxLDL-induced oxidative stress by both direct ROS scavenging and inhibition of NOX activity.     

Protective effects of ellagic and chlorogenic acids against oxidative stress in PC12 cells

Following exposure of differentiated neuronal PC12 cells to either t-BHP, hydrogen peroxide (H₂O₂) or FeSO₄ various kinds of reactive oxygen species (ROS) are generated leading to oxidative injury. The protective effects of two plant polyphenols, ellagic (EC) and chlorogenic acid (CGA), as well as of two metabolites, caffeic acid (CA) and ferulic acid (FA), were investigated in preincubation and coincubation experiments with respect to the following parameters: prevention of cell death, GSH depletion, lipid peroxidation and ROS formation.

The polyphenols more efficiently suppressed cytotoxicity and loss of GSH caused by peroxides than by iron, particularly in preincubation. Lipid peroxidation which increased much stronger in response to FeSO₄ was counteracted completely by the polyphenols. In case of iron, however, only coincubation was effective. EA and CGA and the metabolites CA and FA showed excellent elimination of ROS induced by all stressors. These findings suggest that two dietary antioxidants, EA and CGA, may have protective properties against oxidative stress induced in CNS.     

Dual role of lipoproteins in endothelial cell dysfunction in atherosclerosis

The endothelium is a key constituent of the vascular wall, being actively involved in maintaining the structural integrity and proper functioning of blood vessels. Hyperlipidemia, diabetes, hypertension, smoking and aging are important risk factors for the dysfunction of endothelial cells (EC). Circulating lipoproteins (Lp) synthesized and secreted from the intestine or liver have an important role in supplying peripheral tissues with fatty acids from triglyceride rich lipoproteins (TGRLp) for energy production or storage, and cholesterol from low density lipoproteins (LDL) or high density lipoproteins (HDL) for the synthesis of cellular membranes and steroid hormones. Under pathological conditions, Lp may suffer alterations in concentration and composition and become aggressors for EC. Modified LDL, remnant Lp, TGRLp lipolysis products, dysfunctional HDL are involved in the changes induced in EC morphology (reduced glycocalyx, overdeveloped endoplasmic reticulum, Golgi apparatus and basement membrane), loose intercellular junctions, increased oxidative and inflammatory stress, nitric oxide/redox imbalance, excess Lp transport and storage, as well as loss of anti-thrombotic properties, all of these being characteristics of endothelial dysfunction. Normal HDL are able to counteract the harmful effects of atherogenic Lp in EC but under persistent pathological conditions they lose the protective properties and become pro-atherogenic. This review summarises recent advances in understanding the role of Lp in the induction of endothelial dysfunction and the initiation and progression of atherosclerotic lesions. Its main focus is the antagonistic role of atherogenic Lp (LDL, VLDL, dysfunctional HDL) versus anti-atherogenic Lp (HDL), also pointing out the potential targets for arresting or reversing this process.     

Transport and bioactivity of cyanidin 3-glucoside into the vascular endothelium

Flavonoids are dietary components involved in decreasing oxidative stress in the vascular endothelium and thus the risk of endothelial dysfunction. However, their very low concentrations in plasma place this role in doubt. Thus, a relationship between the effective intracellular concentration of flavonoids and their bioactivity needs to be assessed. This study examined the uptake of physiological concentrations of cyanidin 3-glucoside, a widespread dietary flavonoid, into human vascular endothelial cells. Furthermore, the involvement of the membrane transporter bilitranslocase (TC No. 2.A.65.1.1) as the key underlying molecular mechanism for membrane transport was investigated by using purified anti-sequence antibodies binding at the extracellular domain of the protein. The experimental observations were carried out in isolated plasma membrane vesicles and intact endothelial cells from human endothelial cells (EA.hy926) and on an ischemia-reperfusion model in isolated rat hearts. Cyanidin 3-glucoside was transported via bilitranslocase into endothelial cells, where it acted as a powerful intracellular antioxidant and a cardioprotective agent in the reperfusion phase after ischemia. These findings suggest that dietary flavonoids, despite their limited oral bioavailability and very low postabsorption plasma concentrations, may provide protection against oxidative stress-based cardiovascular diseases. Bilitranslocase, by mediating the cellular uptake of some flavonoids, is thus a key factor in their protective activity on endothelial function.     

Protective effect of prostaglandin A2 against menadione-induced cell injury in cultured porcine aorta endothelial cells

Prostaglandin A2 (PGA2) stimulates the biosynthesis of gamma-glutamylcysteine synthetase and elevates glutathione (GSH) contents in cultured mammalian cells. To clarify the importance of gamma-glutamylcysteine synthetase induction in the defence of endothelial cells against oxidative stress, the effect of PGA2 on menadione (2-methyl-1,4-naphthoquinone)-induced cell injury was examined. Incubation of porcine aorta endothelial cells with menadione produced marked loss of cellular GSH and protein sulfhydryl groups, followed by leakage of lactic dehydrogenase (LDH) into the culture medium. The LDH leakage and modification of protein thiol was, however, completely prevented by pretreatment of the cells with PGA2. The protective effect of PGA2 was more potent than that of cysteine delivery agents such as methionine, N-acetylcysteine or 2-oxo-4-thiazolidine carboxylic acid (OTC). The results suggest that cellular GSH plays an important role in the defence against oxidative stress, and induction of gamma-glutamylcysteine synthetase is effective for protecting vascular endothelial cells.     

Protective Effects of PARP-1 Knockout on Dyslipidemia-Induced Autonomic and Vascular Dysfunction in ApoE?/? Mice: Effects on eNOS and Oxidative Stress

The aims of this study were to investigate the role of poly(ADP-ribose) polymerase (PARP)-1 in dyslipidemia-associated vascular dysfunction as well as autonomic nervous system dysregulation. Apolipoprotein (ApoE)^−/− mice fed a high-fat diet were used as a model of atherosclerosis. Vascular and autonomic functions were measured in conscious mice using telemetry. The study revealed that PARP-1 plays an important role in dyslipidemia-associated vascular and autonomic dysfunction. Inhibition of this enzyme by gene knockout partially restored baroreflex sensitivity in ApoE^−/− mice without affecting baseline heart-rate and arterial pressure, and also improved heart-rate responses following selective blockade of the autonomic nervous system. The protective effect of PARP-1 gene deletion against dyslipidemia-induced endothelial dysfunction was associated with preservation of eNOS activity. Dyslipidemia induced PARP-1 activation was accompanied by oxidative tissue damage, as evidenced by increased expression of iNOS and subsequent protein nitration. PARP-1 gene deletion reversed these effects, suggesting that PARP-1 may contribute to vascular and autonomic pathologies by promoting oxidative tissue injury. Further, inhibition of this oxidative damage may account for protective effects of PARP-1 gene deletion on vascular and autonomic functions. This study demonstrates that PARP-1 participates in dyslipidemia-mediated dysregulation of the autonomic nervous system and that PARP-1 gene deletion normalizes autonomic and vascular dysfunctions. Maintenance of eNOS activity may be associated with the protective effect of PARP-1 gene deletion against dyslipidemia-induced endothelial dysfunction.     

Protectin DX prevents H2O2-mediated oxidative stress in vascular endothelial cells via an AMPK-dependent mechanism

Protectin DX (PDX), which is a novel regulator of 5′ adenosine monophosphate-activated protein kinase (AMPK), has recently gained attention for its ability to improve several metabolic diseases. However, the function of PDX in vascular endothelial cells remains unclear. To confirm the protective effects of PDX on endothelial oxidative stress, human umbilical vein endothelial cells (HUVECs) were treated with hydroperoxide (H₂O₂) and PDX. PDX treatment significantly increased the level of AMPK phosphorylation, and this elevation was attenuated after treatment with G-protein coupled receptor 120 (GPR120) antagonist or GPR120 knockdown. Expressions and activities of antioxidant proteins, including catalase and superoxide dismutase 2 (SOD2), were elevated by PDX and were inhibited by treatment with AMPK inhibitor or with GPR120 antagonist. Production of H₂O₂-induced reactive oxygen species (ROS), the Bax/Bcl-2 ratio, and the loss of mitochondrial membrane potential were all reversed by PDX, leading to improved cell viability and reduced release of lactate dehydrogenase (LDH). Using flow cytometry, we also found that PDX significantly reduced the H₂O₂-induced apoptotic population of cells. These protective effects of PDX were all reversed after treatment with AMPK inhibitor or GRP120 antagonist. These results show that the PDX-AMPK axis has a protective role against H₂O₂-induced oxidative stress in vascular endothelial cells.     

Antioxidant activity of two dibenzocyclooctene lignans on the aged and ischemic brain in rats.

The effects of two dibenzocyclooctene lignans on peroxidative damage of aging and ischemic rat brain were studied. Incubation of eight-month-old rat brain mitochondria and membrane suspension with Fe(2+)-cysteine resulted in the formation of malondialdehyde (MDA) and decrease of ATPase activity. Schisanhenol (Sal) (10(-4) M) completely inhibited the peroxidative damages of brain mitochondria and membrane of rats. The swelling and disintegration of brain mitochondria, as well as the reduction of brain membrane fluidity induced by Fe(2+)-cysteine were also prevented by Sal. The results of imitative experiment of ischemia and reperfusion of brain mitochondria and membrane in vitro indicated that Sal significantly impeded production of MDA and loss of ATPase activity induced by reoxygenation following anoxia. Oral administration of Sal induced increase of cytosol glutathione-peroxidase of brain in mice under the condition of reoxygenation following anoxia. The other compound schizandrin (Sin B) also has similar activity. But its potency is weaker than that of Sal. All these results indicate that Sal and Sin B have protective action against oxidative stress.     

Vitamin E Induces Phosphatidylserine Externalization and Red Cell Adhesion to Endothelial Cells

Red blood cell (RBC) adhesion to vessel wall endothelium is a potent catalyst of vascular occlusion and occurs in oxidative stress states such as hemoglobinopathies and cardiovascular conditions. These are often treated with vitamin E (VitE), a “classic” antioxidant. In this study, we examined the effects of VitE on RBC adhesion to vascular endothelial cells (EC), and on translocation of phosphatidylserine (PS) to RBC surface, known as a potent mediator of RBC/EC adhesion, facilitating thrombus formation. Treatment of RBC with VitE strongly induces (up to sevenfold) PS externalization and enhances (up to 20-fold) their adherence to EC. The VitE hydrophilic analogue—Trolox—does not incorporate into cell membranes. Trolox did not exhibit any of these effects, implying that the VitE effect is due to its known ability to incorporate into cell membranes. The membrane-incorporated VitE significantly reduced the level of reactive oxygen species in H₂O₂-treated RBC, demonstrating that VitE elevates RBC/EC adhesion despite acting as an anti-oxidant. This study demonstrates for the first time that contrary to the common view of VitE as a beneficial supplement, VitE may introduce a circulatory risk by inducing flow-disturbing RBC adherence to blood vessel wall and the pro-thrombotic PS exposure.     

Proteasome inhibition in oxidative stress neurotoxicity: implications for heat shock proteins

Recent studies have demonstrated that inhibition of the proteasome, an enzyme responsible for the majority of intracellular proteolysis, may contribute to the toxicity associated with oxidative stress. In the present study we demonstrate that exposure to oxidative injury (paraquat, H(2)O(2), FeSO(4)) induces a rapid increase in reactive oxygen species (ROS), loss of mitochondrial membrane potential, inhibition of proteasome activity, and induction of cell death in neural SH-SY5Y cells. Application of proteasome inhibitors (MG115, epoxomycin) mimicked the effects of oxidative stressors on mitochondrial membrane potential and cell viability, and increased vulnerability to oxidative injury. Neural SH-SY5Y cells stably transfected with human HDJ-1, a member of the heat shock protein family, were more resistant to the cytotoxicity associated with oxidative stressors. Cells expressing increased levels of HDJ-1 displayed similar degrees of ROS formation following oxidative stressors, but demonstrated a greater preservation of mitochondrial function and proteasomal activity following oxidative injury. Cells transfected with HDJ-1 were also more resistant to the toxicity associated with proteasome inhibitor application. These data support a possible role for proteasome inhibition in the toxicity of oxidative stress, and suggest heat shock proteins may confer resistance to oxidative stress, by preserving proteasome function and attenuating the toxicity of proteasome inhibition.     

Delphinidin-3-Glucoside Protects against Oxidized Low-Density Lipoprotein-Induced Mitochondrial Dysfunction in Vascular Endothelial Cells via the Sodium-Dependent Glucose Transporter SGLT1

Delphinidin-3-glucoside (Dp) is a member of a family of bioactive compounds known as anthocyanins that occur naturally in pigmented plants and are known to ameliorate oxidative stress. Previous studies have showed that Dp decreased oxidative stress in vascular endothelial cells, however, the underlying mechanisms remain largely unknown. In the present study, we showed that pretreatment with Dp significantly suppressed oxidized low-density lipoprotein (oxLDL)-induced cell proliferation inhibition and apoptosis in primary human umbilical vein endothelial cells (HUVECs). Also, Dp pretreatment attenuated oxLDL-induced mitochondrial dysfunction via decreased reactive oxygen species (ROS) and superoxide anion generation, thereby repressing mitochondrial membrane potential and closing mitochondrial permeability transition pore. Furthermore, in vitro and in vivo data showed that Dp was transported into endothelial cells in a temperature, concentration, and time-dependent manner via the sodium-dependent glucose transporter (SGLT1). Suppression of SGLT1 by its substrate glucose, its inhibitor phlorizin or SGLT1 siRNA blocked Dp transportation. Repression of SGLT1 significantly inhibited Dp function of ameliorating mitochondrial dysfunction induced by pro-apoptotic factors (Apoptosis-inducing factor, Cytochrome c, Caspase-3 and Bax/Bcl-2 ratio). Taken together, our data indicate that Dp protects VECs via the SGLT1-ROS-mitochodria pathway. This new insight may help to elucidate the molecular mechanisms underlying the vascular protection afforded by Dp, and anthocyanins in general, in the context of prevention of endothelial dysfunction and atherosclerosis.     

Lung endothelial barrier protection by resveratrol involves inhibition of HMGB1 release and HMGB1-induced mitochondrial oxidative damage via an Nrf2-dependent mechanism

High-mobility group box 1 (HMGB1) contributes to lung vascular hyperpermeability during ventilator-induced lung injury. We aimed to determine whether the natural antioxidant resveratrol protected against HMGB1-induced endothelial hyperpermeability both in vitro and in vivo. We found that HMGB1 decreased vascular endothelial (VE)-cadherin expression and increased endothelial permeability, leading to mitochondrial oxidative damage in primary cultured mouse lung vascular endothelial cells (MLVECs). Both the mitochondrial superoxide dismutase 2 mimetic MnTBAP and resveratrol blocked HMGB1-induced mitochondrial oxidative damage, VE-cadherin downregulation, and endothelial hyperpermeability. In in vivo studies, anesthetized male ICR mice were ventilated for 4 h using low tidal volume (6 ml/kg) or high tidal volume (HV_T; 30 ml/kg) ventilation. The mice were injected intraperitoneally with resveratrol immediately before the onset of ventilation. We found that resveratrol attenuated HV_T-associated lung vascular hyperpermeability and HMGB1 production. HV_T caused a significant increase in nuclear factor-erythroid 2-related factor 2 (Nrf2) nuclear translocation and Nrf2 target gene expression in lung tissues, which was further enhanced by resveratrol treatment. HMGB1 had no effect on Nrf2 activation, whereas resveratrol treatment activated the Nrf2 signaling pathway in HMGB1-treated MLVECs. Moreover, Nrf2 knockdown reversed the inhibitory effects of resveratrol on HMGB1-induced mitochondrial oxidative damage and endothelial hyperpermeability. The inhibitory effect of resveratrol on cyclic stretch-induced HMGB1 mRNA expression in primary cultured MLVECs was also abolished by Nrf2 knockdown. In summary, this study demonstrates that resveratrol protects against lung endothelial barrier dysfunction initiated by HV_T. Lung endothelial barrier protection by resveratrol involves inhibition of mechanical stretch-induced HMGB1 release and HMGB1-induced mitochondrial oxidative damage. These protective effects of resveratrol might be mediated through an Nrf2-dependent mechanism.     

Brain Iron Toxicity: Differential Responses of Astrocytes,Neurons, and Endothelial Cells

Iron accumulation or iron overload in brain is commonly associated with neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases, and also plays a role in cellular damage following hemorrhagic stroke and traumatic brain injury. Despite the brain’s highly regulated system for iron utilization and metabolism, these disorders often present following disruptions within iron metabolic pathways. Such dysregulation allows saturation of proteins involved in iron transport and storage, and may cause an increase in free ferrous iron within brain leading to oxidative damage. Not only do astrocytes, neurons, and brain endothelial cells serve unique purposes within the brain, but their individual cell types are equipped with distinct protective mechanisms against iron-induced injury. This review evaluates iron metabolism within the brain under homeostatic and pathological conditions and focuses on the mechanism(s) of brain cellular iron toxicity and differential responses of astrocytes, neurons, and brain vascular endothelial cells to excessive free iron. Special issue dedicated to Dr. Moussa Youdim. An erratum to this article can be found at     

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.