Platelets enhance endothelial adhesiveness in high tidal volume ventilation

Although platelets induce lung inflammation, leading to acute lung injury (ALI), the extent of platelet-endothelial cell (EC) interactions remains poorly understood. Here, in a ventilation-stress model of lung inflammation, we show that platelet-EC interactions are important. We obtained freshly isolated lung endothelial cells (FLECs) from isolated, blood-perfused rat lungs exposed to ventilation at low tidal volume (LV) or stress-inducing high tidal volume (HV). Immunofluorescence and immunoprecipitation studies revealed HV-induced increases in cell-surface von Willebrand factor (vWf) expression on FLEC. This increased expression was inhibited by platelet removal from the lung perfusion and by including a P-selectin-blocking antibody in the lung perfusion. The expression was also blocked in lungs from P-selectin knockout (P sel(-/-)) mice perfused with autologous blood, but not with heterologous wild-type blood containing P-selectin-expressing platelets. These findings indicate that in ventilation stress, platelets transfer vWf to the EC surface and that platelet P-selectin plays a critical role in this transfer. Further evidence for such intercellular transfers was the HV-induced FLEC expressions of platelet glycoprotein 1b and of platelet P-selectin. We conclude that in ventilation stress, platelets deposit leukocyte- and platelet-binding proteins on the EC surface, thereby establishing the proinflammatory phenotype of the vascular lining.     

Sequence of endothelial signaling during lung expansion

Although high tidal volume ventilation exacerbates lung injury, the mechanisms underlying the inflammatory response are not clear. Here, we exposed isolated lungs to high or low tidal volume ventilation, while perfusing lungs with whole blood, or blood depleted of leukocytes and platelets. Then, we determined signaling responses in freshly isolated lung endothelial cells by means of immunoblotting and immunofluorescence approaches. In depleted blood perfusion, high tidal volume induced modest increases in both P-selectin expression on the endothelial surface, and in endothelial protein tyrosine phosphorylation. Both high tidal volume-induced responses were markedly enhanced in the presence of whole blood perfusion. However, a P-selectin-blocking antibody given together with whole blood perfusion inhibited the responses down to levels corresponding to those for depleted blood perfusion. These findings indicate that the full proinflammatory response occurs in two stages. First, lung distension causes modest endothelial activation. Second, subsequent endothelial-inflammatory cell interactions augment P-selectin expression and tyrosine phosphorylation. We conclude that interactions of circulating inflammatory cells with P-selectin critically determine proinflammatory endothelial activation during high tidal volume ventilation.     

Paxillin is involved in the differential regulation of endothelial barrier by HGF and VEGF

Circulating levels of hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) are increased during acute lung injury; however, combined effects of HGF and VEGF on pulmonary endothelial cell (EC) permeability remain to be elucidated. We have previously shown differential remodeling of focal adhesions (FA) caused by barrier-protective and barrier-disruptive mechanical and chemical stimuli. This study examined a role of FA protein paxillin in the pulmonary EC barrier responses induced by HGF and VEGF. VEGF increased, but HGF decreased, pulmonary EC permeability. These effects were accompanied by differential patterns of site-specific phosphorylation of focal adhesion kinase (FAK) and paxillin and FA redistribution. HGF antagonized random FA formation caused by VEGF challenge and promoted FA accumulation at the cell periphery. HGF attenuated VEGF-induced paxillin redistribution, FA remodeling, and endothelial permeability. SiRNA-based paxillin knockdown attenuated VEGF-induced EC permeability, myosin light chain phosphorylation, and stress fiber and paracellular gap formation. Paxillin knockdown also decreased HGF-induced EC barrier enhancement and suppressed activation of Rac and its effector PAK1. Expression of paxillin-S(273) deficient on PAK1 phosphorylation site prevented HGF-induced cytoskeletal remodeling. These data show a dual role of paxillin in the HGF- and VEGF-mediated endothelial barrier regulation and suggest essential paxillin role in the modulation of Rac-Rho crosstalk. Our results also support a model of pulmonary EC barrier recovery during resolution of ALI via switch from VEGF to HGF signaling.     

Stable lymphocyte contact induces remodeling of endothelial cell matrix receptor complexes

Endothelial cells (EC) actively participate in lymphocyte transendothelial migration by remodeling their actin cytoskeleton. We studied the endothelial cell abluminal matrix receptor (focal adhesion, FA) complexes to determine if these structures were remodeled following lymphocyte adhesion. Lymphocytes (PBL) were isolated from whole blood and added to cultured EC. Lectin-stimulated PBL adhered to EC spontaneously, whereas adhesion of freshly isolated lymphocytes to EC was induced by pre-treatment with MCP-1 or activating anti-CD11a mAb. Sustained adhesion between lymphocytes and EC resulted in a significant, contact-dependent decrease in paxillin incorporation into the FA following 15, but not 5, min of contact. EC FA remodeling was associated with increased phosphorylation of pp125 FA kinase. Pretreatment of the EC with an activating beta1 integrin monoclonal antibody, TS2/16, prevented lymphocyte-stimulated FA remodeling. Further, TS2/16 pretreatment inhibited transendothelial migration of lymphocytes and beta1 integrin-deficient JY lymphoblasts. These data demonstrate that sustained lymphocyte adhesion induces remodeling of EC FA structures and that this remodeling event is required for efficient lymphocyte transendothelial migration in vitro.     

Exogenous Nitric Oxide Increases Neutrophil Adhesion to Cultured Human Endothelial Monolayers through a Protein Kinase G Dependent Mechanism

Endothelial-neutrophil adhesion is a critical step in acute inflammatory diseases, which is mediated in part by P-selectin and platelet-activating factor (PAF). Nitric oxide (NO) is well known as an endogenous second messenger derived from endothelial cells, and regulates many important physiological events, however, the direct effects of NO on endothelial-neutrophil adhesion is less well understood. The objective of this study was to examine whether, and how relatively high levels of exogenous NO increases neutrophil adhesion with respect to P-selectin and PAF. Endothelial monolayers were exposed to chemical agents for 30 min, and the adhesion of ^5lCr-labeled neutrophils measured in a static adhesion assay. Spermine-NONOate (SNO), an NO donor, significantly increased neutrophil adhesion and expression of P-selectin at a concentration of 1 mM. SNO (1 mM)-mediated neutrophil adhesion was significantly inhibited by a protein kinase G inhibitor, KT5823 (0.5 M), but not by a classical protein kinase C inhibitor, Gö6976 (10 nM), a tyrosine kinase inhibitor, genistein (1 M), or a protein kinase A inhibitor, H-89 (0.1 M). P-selectin surface expression induced by 1 mM SNO was also significantly inhibited by 0.5 M KT5823. Conversely, a cytoplasm calcium chelator, TMB-8 (0.1 mM), significantly exacerbated both the neutrophil adhesion and P-selectin expression induced by SNO. WEB 2086 (10 M), a PAF receptor antagonist, blocked neutrophil adhesion, but did not block P-selectin expression induced by SNO. These data suggest that NO increases endothelial-neutrophil adhesion through protein kinase G-mediated P-selectin mobilization to the cell surface and endothelial PAF synthesis.     

Angiopoietin-mediated endothelial P-selectin translocation: cell signaling mechanisms

Recently identified, angiopoietin-1 (Ang1) and -2 (Ang2) bind to the tyrosine kinase receptor Tie2 and contribute to orchestrate blood vessel formation during angiogenesis. Ang1 mediates vessel maturation and integrity by favoring the recruitment of pericytes and smooth muscle cells. Ang2, initially identified as a Tie2 antagonist, may under certain circumstances, induce Tie2 phosphorylation and biological activities. As inflammation exists in a mutually dependent association with angiogenesis, we sought to determine if Ang1 and/or Ang2 could modulate proinflammatory activities, namely P-selectin translocation, in bovine aortic endothelial cells (EC) and dissect the mechanisms implicated. P-selectin, an adhesion molecule found in the Weibel-Palade bodies of EC, is translocated rapidly to the cell surface upon EC activation during inflammatory processes. Herein, we report that Ang1 and Ang2 (1 nM) are capable of mediating a rapid Tie2 phosphorylation as well as a rapid and transient endothelial P-selectin translocation maximal within 7.5 min (125% and 100% increase, respectively, over control values). In addition, we demonstrate for the first time that angiopoietin-mediated endothelial P-selectin translocation is calcium-dependent and regulated through phospholipase C-gamma activation.     

Candida albicans expresses a focal adhesion kinase-like protein that undergoes increased tyrosine phosphorylation upon yeast cell adhesion to vitronectin and the EA.hy 926 human endothelial cell line

The signaling pathways triggered by adherence of Candida albicans to the host cells or extracellular matrix are poorly understood. We provide here evidence in C. albicans yeasts of a p105 focal adhesion kinase (Fak)-like protein (that we termed CaFak), antigenically related to the vertebrate p125Fak, and its involvement in integrin-like-mediated fungus adhesion to vitronectin (VN) and EA.hy 926 human endothelial cell line. Biochemical analysis with different anti-chicken Fak antibodies identified CaFak as a 105-kDa protein and immunofluorescence and cytofluorimetric analysis on permeabilized cells specifically stain C. albicans yeasts; moreover, confocal microscopy evidences CaFak as a cytosolic protein that colocalizes on the membrane with the integrin-like VN receptors upon yeast adhesion to VN. The protein tyrosine kinase (PTK) inhibitors genistein and herbimycin A strongly inhibited C. albicans yeast adhesion to VN and EA.hy 926 endothelial cells. Moreover, engagement of alpha v beta 3 and alpha v beta 5 integrin-like on C. albicans either by specific monoclonal antibodies or upon adhesion to VN or EA.hy 926 endothelial cells stimulates CaFak tyrosine phosphorylation that is blocked by PTK inhibitor. A role for CaFak in C. albicans yeast adhesion was also supported by the failure of VN to stimulate its tyrosine phosphorylation in a C. albicans mutant showing normal levels of CaFak and VNR-like integrins but displaying reduced adhesiveness to VN and EA.hy 926 endothelial cells. Our results suggest that C. albicans Fak-like protein is involved in the control of yeast cell adhesion to VN and endothelial cells.     

Hypoxia-induced mitogenic factor promotes vascular adhesion molecule-1 expression via the PI-3K/Akt-NF-kappaB signaling pathway

Hypoxia-induced mitogenic factor (HIMF), also known as FIZZ1 (found in inflammatory zone 1), is an important player in lung inflammation. However, the effects of HIMF on cell adhesion molecules involved in lung inflammation remain largely unknown. In the present work, we tested whether HIMF modulates vascular adhesion molecule (VCAM)-1 expression, and dissected the possible signaling pathways that link HIMF to VCAM-1 upregulation. Recombinant HIMF protein, instilled intratracheally into adult mouse lungs, results in a significant increase of VCAM-1 production in vascular endothelial, alveolar type II, and airway epithelial cells. In cultured mouse endothelial SVEC 4-10 and lung epithelial MLE-12 cells, we demonstrated that HIMF induces VCAM-1 expression via the phosphatidylinositol-3 kinase (PI-3K)/Akt-nuclear factor (NF)-kappaB signaling pathway. Knockdown of HIMF expression by small interference RNA attenuated LPS-induced VCAM-1 expression in vitro. We showed that HIMF induced phosphorylation of the IkappaB kinase signalsome and, subsequently, IkappaBalpha, leading to activation of NF-kappaB. Meanwhile, VCAM-1 production was correspondingly upregulated. Blocking NF-kappaB signaling pathway by expression of dominant-negative mutants of IkappaB kinase and IkappaBalpha suppressed HIMF-induced VCAM-1 upregulation. HIMF also strongly induced phosphorylation of Akt. A dominant-negative mutant of PI-3K, Deltap85, as well as PI-3K inhibitor, LY294002, also blocked HIMF-induced NF-kappaB activation and attenuated VCAM-1 production. Furthermore, LY294002 pretreatment abolished HIMF-enhanced mononuclear cells adhesion to endothelial and epithelial cells. Our findings connect HIMF to signaling pathways that regulate inflammation, and thus reveal the critical roles that HIMF plays in lung inflammation.     

Modulation of prosurvival signaling in fibroblasts by a protein kinase inhibitor protects against fibrotic tissue injury

Progressive fibrotic diseases involving diverse organ systems are associated with the persistence of fibroblasts/myofibroblasts in injured tissues. Activation of focal adhesion kinase (FAK) and protein kinase B (PKB/Akt) by transforming growth factor-beta1 mediate stable induction of myofibroblast differentiation and survival. In this report, we demonstrate that transforming growth factor-beta1-induced activation of both PKB/Akt and FAK are dose dependently inhibited by the protein kinase inhibitor, AG1879, in cultured human lung fibroblasts. In a murine model of intratracheal bleomycin-induced lung fibrosis, regions of active fibrogenesis demonstrate elevated expression of PKB/Akt and FAK phosphorylation in vivo, effects that are attenuated in mice receiving daily intraperitoneal injections of AG1879 (bleomycin-AG1879) versus a chemically inactive analog (bleomycin-control). PKB/Akt and FAK phosphorylation are elevated in fibroblasts isolated from lungs of bleomycin-injured mice, effects that are inhibited in bleomycin-AG1879 mice. Accumulation of alpha-smooth muscle actin-expressing myofibroblasts is markedly reduced in lungs of bleomycin-AG1879 mice. The numbers of recruited inflammatory cells were not significantly different between these groups. Bleomycin-AG1879 mice are protected from lung fibrosis as evidenced by histopathology, trichrome staining, and biochemical analysis for collagen. Thus, targeting of prosurvival signaling pathways in fibroblasts/myofibroblasts may provide a novel and effective strategy for anti-fibrotic therapy of treatment-unresponsive fibrotic disorders.     

Hyperoxia-induced reactive oxygen species formation in pulmonary capillary endothelial cells in situ

Lung capillary endothelial cells (ECs) are a critical target of oxygen toxicity and play a central role in the pathogenesis of hyperoxic lung injury. To determine mechanisms and time course of EC activation in normobaric hyperoxia, we measured endothelial concentration of reactive oxygen species (ROS) and cytosolic calcium ([Ca(2+)](i)) by in situ imaging of 2',7'-dichlorofluorescein (DCF) and fura 2 fluorescence, respectively, and translocation of the small GTPase Rac1 by immunofluorescence in isolated perfused rat lungs. Endothelial DCF fluorescence and [Ca(2+)](i) increased continuously yet reversibly during a 90-min interval of hyperoxic ventilation with 70% O(2), demonstrating progressive ROS generation and second messenger signaling. ROS formation increased exponentially with higher O(2) concentrations. ROS and [Ca(2+)](i) responses were blocked by the mitochondrial complex I inhibitor rotenone, whereas inhibitors of NAD(P)H oxidase and the intracellular Ca(2+) chelator BAPTA predominantly attenuated the late phase of the hyperoxia-induced DCF fluorescence increase after > 30 min. Rac1 translocation in lung capillary ECs was barely detectable at normoxia but was prominent after 60 min of hyperoxia and could be blocked by rotenone and BAPTA. We conclude that hyperoxia induces ROS formation in lung capillary ECs, which initially originates from the mitochondrial electron transport chain but subsequently involves activation of NAD(P)H oxidase by endothelial [Ca(2+)](i) signaling and Rac1 activation. Our findings demonstrate rapid activation of ECs by hyperoxia in situ and identify mechanisms that may be relevant in the initiation of hyperoxic lung injury.     

CNTO 95, a fully human anti αv integrin antibody, inhibits cell signaling, migration, invasion, and spontaneous metastasis of human breast cancer cells

CNTO 95 is a fully human monoclonal antibody that recognizes αv integrins. Previous studies have shown that CNTO 95 exhibits both anti-tumor and anti-angiogenic activities (Trikha M et al., Int J Cancer 110:326–335, 2004). In this study we investigated the biological activities of CNTO 95 on breast tumor cells both in vitro and in vivo. In vitro treatment with CNTO 95 decreased the viability of breast tumor cells adhering to vitronectin. CNTO 95 inhibited tumor cell adhesion, migration, and invasion in vitro. CNTO 95 treatment also induced tyrosine dephosphorylation of focal adhesion kinase (FAK), and the docking protein paxillin that recruits both structural and signaling molecules to focal adhesions (Turner CE, Int J Biochem Cell Biol 30:955–959, 1998; O’Neil GM et al., Trends Cell Biol 10:111–119, 2000). These results suggest that CNTO 95 inhibits breast tumor cell growth, migration and invasion by interruption of αv integrin mediated focal adhesions and cell motility signals. In vivo studies of CNTO 95 were conducted in an orthotopic breast tumor xenograft model. Treatment with CNTO 95 resulted in significant inhibition of both tumor growth and spontaneous metastasis of MDA-MB-231 cells to the lungs. CNTO 95 also inhibited lung metastasis in a separate experimental (tail vein injection) model of metastasis. The results presented here demonstrate the anti-tumor and anti-metastatic activities of CNTO 95 in breast cancer models and provide insight into the cellular and molecular mechanisms mediating its inhibitory effects on metastasis.     

Neutrophil Adhesion to Human Endothelial Cells is Induced by the Membrane Attack Complex: The Roles of P-Selectin and Platelet Activating Factor

A variety of inflammatory diseases are accompanied by activation of the complement system. We examined the role of the membrane attack complex (MAC) in mediating neutrophil adhesion to endothelial cells. To assemble the MAC in endothelial cell monolayers, a C5b-like molecule was created through the treatment of purified C5 with the oxidizing agent chloramine-T, followed by addition of the remaining components (C6-C9) that constitute the MAC. Use of this method abrogated potentially confounding effects mediated by other complement components (e.g., C5a). MAC assembly resulted in a rapid (30 min), concentration-dependent increase in neutrophil adherence. A monoclonal antibody directed against P-selectin inhibited MAC-mediated neutrophil adhesion. A whole cell EIA confirmed P-selectin expression after formation of the MAC. Incubation of neutrophils with the platelet-activating factor receptor antagonist, CF 3988, also significantly decreased adhesion, indicating that PAF plays a role in MAC-mediated adhesion. These results suggest that the MAC can promote neutrophil adhesion through P-selectin and PAF-mediated mechanisms.     

Schistosoma mansoni schistosomula reduce E-selectin and VCAM-1 expression in TNF-alpha-stimulated lung microvascular endothelial cells by interfering with the NF-kappaB pathway

The recruitment of immune cells into the lungs is a key step in protection against murine schistosomiasis. In this phenomenon, pulmonary (micro)vascular endothelial cells (EC) probably play a central role, by expressing specific adhesion molecules on their surface. Recently, we have shown that Schistosoma mansoni schistosomula, the parasitic stage which resides in the lungs, could activate microvascular EC to acquire an anti-inflammatory phenotype. In the present study, we tested the hypothesis that schistosomula could also regulate the expression of adhesion molecules in vitro by human lung microvascular EC (HMVEC-l) in the present of the pro-inflammatory cytokine TNF-alpha. We found that lipophilic substance(s) present in the excretory/secretory products from schistosomula selectively reduce the TNF-alpha-induced synthesis of E-selectin and VCAM-1 mRNA and proteins without affecting ICAM-1. This inhibitory effect appears to be mediated by a cyclic AMP/protein kinase A (cAMP/PKA) pathway that probably interferes with the NF-kappaB pathway induced by TNF-alpha at the level of the E-selectin promoter, whereas a cAMP-independent pathway appears to operate in VCAM-1 down-modulation. Finally, schistosomula also significantly reduce the VLA-4/VCAM-1-dependent adherence of leukocytes to TNF-alpha-stimulated HMVEC-l. We speculate that this mechanism could represent a new stratagem that parasites may use to escape the immune system by controlling leukocyte recruitment to the lungs.     

Focal adhesion kinase is involved in type III group B streptococcal invasion of human brain microvascular endothelial cells

Group B streptococcus (GBS), the leading cause of neonatal meningitis, has been shown to invade human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. GBS invasion of HBMEC has been shown to require the host cell actin cytoskeleton rearrangements. The present study examined the mechanisms underlying actin cytoskeleton rearrangements that are involved in type III GBS invasion of HBMEC. We showed that type III GBS invasion was inhibited by genistein, a general tyrosine kinase inhibitor (mean 54% invasion decrease at 100 microM), and LY294002, a phosphatidylinositol 3 (PI3) kinase inhibitor (mean 70% invasion decrease at 50 microM), but not by PP2, an inhibitor of the Src family tyrosine kinases. We subsequently showed that the focal adhesion kinase (FAK) was the one of the host proteins tyrosine phosphorylated by type III GBS. Over-expression of a dominant negative form of the FAK C-terminal domain significantly decreased type III GBS invasion of HBMEC (mean 51% invasion decrease). In addition, we showed that FAK phosphorylation correlated with its association of paxillin, an adapter protein of actin filament, and PI3-kinase subunit p85. This is the first demonstration that FAK phosphorylation and its association with paxillin and PI3 kinase play a key role in type III GBS invasion of HBMEC.     

Vitamin E inhibits lysophosphatidylcholine-induced endothelial dysfunction and platelet activation

Lysophosphatidylcholine (LPC), a lysolipid contained in oxidized low-density lipoprotein, is an atherogenic molecule that induces endothelial dysfunction and platelet activation and inhibits angiogenesis. Although studies showed that vitamin E has antiatherogenic properties, the effects of vitamin E on LPC-induced endothelial dysfunction and platelet activation are little known. We examined whether vitamin E has protecting actions against LPC-induced alterations of endothelial and platelet functions. Incubation of cultured bovine aortic endothelial cells (BAECs) with LPC (10 microM) significantly inhibited bradykinin (1 microM)-stimulated nitric oxide release, which was prevented by cotreatment with vitamin E (50, 100, and 500 microg/ml) in a concentration-dependent manner. In isolated human platelets, LPC stimulated P-selectin expression and induced leukocyte-platelet interaction, which functionally depends on P-selectin expressed on the platelet surface. Vitamin E treatment significantly prevented the LPC-induced platelet P-selectin expression and leukocyte-platelet interaction. As LPC-induced endothelial dysfunction and platelet activation have been shown to involve the protein kinase C (PKC)-dependent signal transduction pathway, we examined the effects of vitamin E on LPC-induced PKC activation in human platelets and BAECs. Vitamin E significantly inhibited LPC (10 microM)-stimulated PKC activation in a concentration-dependent manner. It is concluded that (a) Vitamin E prevented LPC-induced endothelial dysfunction and preserved endothelial nitric oxide release, (b) vitamin E inhibited LPC-induced platelet activation (P-selectin expression) and leukocyte-platelet interaction, and (c) these mechanisms appeared to be at least partly mediated by suppression of the PKC in endothelial cells and platelets. The present findings may provide new insights into antiatherogenic mechanisms of vitamin E.     

The role of scaffold microarchitecture in engineering endothelial cell immunomodulation

The implantation of matrix-embedded endothelial cells (MEECs) has been reported to have great therapeutic potential in controlling the vascular response to injury and maintaining patency in arteriovenous anastomoses. While there is an appreciation of their effectiveness in clinical and animal studies, the mechanisms through which they mediate these powerful effects remain relatively unknown. In this work, we examined the hypothesis that the 3-dimensional microarchitecture of the tissue engineering scaffold was a key regulator of endothelial behavior in MEEC constructs. Notably, we found that ECs in porous collagen scaffold had a markedly altered cytoskeletal structure with oriented actin fibers and rearrangement of the focal adhesion proteins in comparison to cells grown on 2D surfaces. We examined the immunomodulatory capabilities of MEECs and discovered that they were able to reduce the recruitment of monocytes to an inflamed endothelial monolayer by 5-fold compared to EC on 2D surfaces. An analysis of secreted factors from the cells revealed an 8-fold lower release of Monocyte Chemotactic Protein-1 (MCP-1) from MEECs. Differences between 3D and 2D cultured cells were abolished in the presence of inhibitors to the focal adhesion associated signaling molecule Src suggesting that adhesion-mediated signaling is essential in controlling the potent immunomodulatory effects of MEEC.     

Endothelial paxillin and focal adhesion kinase (FAK) play a critical role in neutrophil transmigration

During an inflammatory response, endothelial cells undergo morphological changes to allow for the passage of neutrophils from the blood vessel to the site of injury or infection. Although endothelial cell junctions and the cytoskeleton undergo reorganization during inflammation, little is known about another class of cellular structures, the focal adhesions. In this study, we examined several focal adhesion proteins during an inflammatory response. We found that there was selective loss of paxillin and focal adhesion kinase (FAK) from focal adhesions in proximity to transmigrating neutrophils; in contrast the levels of the focal adhesion proteins β1-integrin and vinculin were unaffected. Paxillin was lost from focal adhesions during neutrophil transmigration both under static and flow conditions. Down-regulating endothelial paxillin with siRNA blocked neutrophil transmigration while having no effect on rolling or adhesion. As paxillin dynamics are regulated partly by FAK, the role of FAK in neutrophil transmigration was examined using two complementary methods. siRNA was used to down-regulate total FAK protein while dominant-negative, kinase-deficient FAK was expressed to block FAK signaling. Disruption of the FAK protein or FAK signaling decreased neutrophil transmigration. Collectively, these findings reveal a novel role for endothelial focal adhesion proteins paxillin and FAK in regulating neutrophil transmigration.     

Antibody binding to cell surface amyloid precursor protein induces neuronal injury by deregulating the phosphorylation of focal adhesion signaling related proteins

The biological function of full-length amyloid-β protein precursor (APP), the precursor of Aβ, is not fully understood. Mounting studies reported that antibody binding to cell surface APP causes neuronal injury. However, the mechanism of cell surface APP mediating neuronal injury remains to be determined. Colocalization of APP with integrin on cell surface leads us to suppose that focal adhesion (FA) related mechanism is involved in surface APP-mediated neuronal injury. In the present study, results demonstrated that primary cultured neurons treated with antibody against APP-N-terminal not only caused neuronal injury and aberrant morphologic changes of neurite, but also induced reaction of FA proteins appearing an acute increase then decrease pattern. Moreover, the elevation of tyrosine phosphorylation of FA proteins including paxillin and focal adhesion kinase (FAK), and down-regulated expression of protein tyrosine phosphatase (PTP1B) induced by APP antibody were prevented by inhibitor of Src protein kinases 4-amino-5-(4-chlorophenyl)-7(t-butyl) pyrazol (3,4-D) pyramide (PP2) and G protein inhibitor pertussis toxin (PTX), implying that Src family kinase and G protein play roles in APP-induced FA signals. In addition, pretreatment with PTX and PP2 was able to suppress APP-antibody induced neuronal injury. Taken together, the results suggest a novel mechanism for APP mediating neuronal injury through deregulating FA signals.