Intercellular tight junctions (TJs) exhibit a complex molecular architecture involving the regulated cointeraction of cytoplasmic adaptor proteins (e.g., zonula occludens) and integral membrane linker proteins (e.g., occludin and claudins). They provide structural integrity to epithelial and endothelial tissues and create highly polarized barriers essential to homeostatic maintenance within vertebrate physiological systems, while their dysregulation is an established pathophysiological hallmark of many diseases (e.g., cancer, stroke, and inflammatory lung disease). The junctional complex itself is a highly dynamic signaling entity wherein participant proteins constantly undergo a blend of regulatory modifications in response to diverse physiological and pathological cues, ultimately diversifying the overall adhesive properties of the TJ. Occludin, a 65-kDa tetraspan integral membrane protein, contributes to TJ stabilization and optimal barrier function. This paper reviews our current knowledge of how tissue occludin is specifically modified at the posttranscriptional and posttranslational levels in diverse circumstances, with associated consequences for TJ dynamics and epithelial/endothelial homeostasis. Mechanistic concepts such as splice variance and alternate promoter usage, proteolysis, phosphorylation, dimerization, and ubiquitination are comprehensively examined, and possible avenues for future investigation highlighted. (34), is an intramembrane multiprotein complex that provides apical intercelluar connections between adjacent cells in both epithelial and endothelial monolayers. Working in concert with other structurally distinct intercellular junctions (adherens junctions, gap junctions, and desmosomes), TJs provide structural integrity to tissues and create highly polarized barriers with selective paracellular permeability to water, solutes, larger molecules, and other cells, an essential feature of homeostatic maintenance within vertebrate physiological systems. Over the years, additional roles for TJs in cellular differentiation, proliferation, migration, signal transduction, and gene expression have also emerged, highlighting their functional diversity (for reviews, see references 10, 73, and 106), while TJ dysregulation has been associated with the pathogenesis of various diseases, including cancers, stroke, diabetic retinopathy, pulmonary disorders, and inflammatory bowel disease (38). TIGHT JUNCTIONS AND OCCLUDIN T ight junctions. The tight junction (TJ), originally identified in the early 1960s by Farquhar and PaladeThe molecular architecture of the TJ exhibits a complex arrangement of cointeracting cytoplasmic adaptor proteins (e.g., zonula occludens [ZO-1, ZO-2, and ZO-3], as well as 7H6, AF6, vinculin, and cingulin), which mediate the cytoskeletal tethering and cell-cell partnering of transmembrane linker proteins (e.g., occludin, claudins, and junctional adhesion molecules 1, 2, and 3) (1). The overall junctional complex is therefore a highly dynamic signaling entity in which the individual ...

Background and ObjectivesBlood-brain barrier (BBB) dysfunction is an integral feature of neurological disorders and involves the action of multiple proinflammatory cytokines on the microvascular endothelial cells lining cerebral capillaries. There is still however, considerable ambiguity throughout the scientific literature regarding the mechanistic role(s) of cytokines in this context, thereby warranting a comprehensive in vitro investigation into how different cytokines may cause dysregulation of adherens and tight junctions leading to BBB permeabilization.MethodsThe present study employs human brain microvascular endothelial cells (HBMvECs) to compare/contrast the effects of TNF-α and IL-6 on BBB characteristics ranging from the expression of interendothelial junction proteins (VE-cadherin, occludin and claudin-5) to endothelial monolayer permeability. The contribution of cytokine-induced NADPH oxidase activation to altered barrier phenotype was also investigated.ResultsIn response to treatment with either TNF-α or IL-6 (0–100 ng/ml, 0–24 hrs), our studies consistently demonstrated significant dose- and time-dependent decreases in the expression of all interendothelial junction proteins examined, in parallel with dose- and time-dependent increases in ROS generation and HBMvEC permeability. Increased expression and co-association of gp91 and p47, pivotal NADPH oxidase subunits, was also observed in response to either cytokine. Finally, cytokine-dependent effects on junctional protein expression, ROS generation and endothelial permeability could all be attenuated to a comparable extent using a range of antioxidant strategies, which included ROS depleting agents (superoxide dismutase, catalase, N-acetylcysteine, apocynin) and targeted NADPH oxidase blockade (gp91 and p47 siRNA, NSC23766).ConclusionA timely and wide-ranging investigation comparing the permeabilizing actions of TNF-α and IL-6 in HBMvECs is presented, in which we demonstrate how either cytokine can similarly downregulate the expression of interendothelial adherens and tight junction proteins leading to elevation of paracellular permeability. The cytokine-dependent activation of NADPH oxidase leading to ROS generation was also confirmed to be responsible in-part for these events.

Martin FA, Murphy RP, Cummins PM. Thrombomodulin and the vascular endothelium: insights into functional, regulatory, and therapeutic aspects.

Abstract-Notch signaling has been shown recently to regulate vascular cell fate in adult cells. By applying a uniform equibiaxial cyclic strain to vascular smooth muscle cells (SMCs), we investigated the role of strain in modulating Notch-mediated growth of SMCs in vitro. Rat SMCs cultured under conditions of defined equibiaxial cyclic strain (0% to 15% stretch; 60 cycles/min; 0 to 24 hours) exhibited a significant temporal and force-dependent reduction in Notch 3 receptor expression, concomitant with a significant reduction in Epstein Barr virus latency C promoter-binding factor-1/recombination signal-binding protein of the J immunoglobulin gene-dependent Notch target gene promoter activity and mRNA levels when compared with unstrained controls. The decrease in Notch signaling was Gi-proteinand mitogen-activated protein kinase-dependent. In parallel cultures, cyclic strain inhibited SMC proliferation (cell number and proliferating cell nuclear antigen expression) while significantly promoting SMC apoptosis (annexin V binding, caspase-3 activity and bax/bcl-x L ratio). Notch 3 receptor overexpression significantly reversed the straininduced changes in SMC proliferation and apoptosis to levels comparable to unstrained control cells, whereas Notch inhibition further potentiated the changes in SMC apoptosis and proliferation. These findings suggest that cyclic strain inhibits SMC growth while enhancing SMC apoptosis, in part, Key Words: notch Ⅲ cyclic strain Ⅲ apoptosis Ⅲ proliferation Ⅲ vascular Ⅲ G-proteins H emodynamic forces associated with the flow of blood play an important role in the physiological control of vascular tone, remodeling, and associated vascular pathologies. These forces include cyclic circumferential strain, which is caused by a transmural force acting perpendicular to the vessel wall. [1][2][3][4] Mechanotransduction is known to play a central role in the highly coordinated cellular response of the vasculature to changes in hemodynamic stimulation. Transduction of biomechanical stimuli leads to activation of cellular signaling mechanisms that ultimately lead to adaptive, and sometimes maladaptive, changes in cell and tissue fate. 5,6 The ultimate arbiter of vascular cell fate (growth, migration, differentiation, and apoptosis) in response to hemodynamic stimulation is unclear but considered fundamental to the pathogenesis of vascular disease. Strain-induced changes in smooth muscle cell (SMC) growth, defined as the balance between SMC proliferation and apoptosis, participates in the local vascular reaction to hypertension, 3,7 late lumen loss, and restenosis after vascular interventions, as well as plaque vulnerability during athersosclerosis. 1,8 Because changes in vascular cell fate are also apparent during vascular morphogenesis and modeling of the embryonic vasculature, 9,10 the control of these cell fate decisions in adult cells may share similar signaling patterns. Notch receptor-ligand interactions are a highly conserved mechanism, originally described in developmental studies using...

Vascular smooth muscle cell (SMC) fate decisions (cell growth, migration, and apoptosis) are fundamental features in the pathogenesis of vascular disease. We investigated the role of Notch 1 and 3 receptor signaling in controlling adult SMC fate in vitro by establishing that hairy enhancer of split (hes-1 and -5) and related hrt's (hrt-1, -2, and -3) are direct downstream target genes of Notch 1 and 3 receptors in SMC and identified an essential role for nuclear protein CBF-1/RBP-Jk in their regulation. Constitutive expression of active Notch 1 and 3 receptors (Notch IC) resulted in a significant up-regulation of CBF-1/RBP-Jk-dependent promoter activity and Notch target gene expression concomitant with significant increases in SMC growth while concurrently inhibiting SMC apoptosis and migration. Moreover, inhibition of endogenous Notch mediated CBF-1/RBP-Jk regulated gene expression with a non-DNA binding mutant of CBF-1, a Notch IC deleted of its delta RAM domain and the Epstein-Barr virus encoded RPMS-1, in conjunction with pharmacological inhibitors of Notch IC receptor trafficking (brefeldin A and monensin), resulted in a significant decrease in cell growth while concomitantly increasing SMC apoptosis and migration. These findings suggest that endogenous Notch receptors and downstream target genes control vascular cell fate in vitro. Notch signaling, therefore, represents a novel therapeutic target for disease states in which changes in vascular cell fate occur in vivo.

An intact functioning blood-brain barrier (BBB) is fundamental to proper homoeostatic maintenance and perfusion of the central nervous system (CNS). Inflammatory damage to the unique microvascular endothelial cell monolayer that constitutes the luminal BBB surface, leading to elevated capillary permeability, has been linked to various neurological disorders ranging from ischaemic stroke and traumatic brain injury, to neurodegenerative disease and CNS infections. Moreover, the neuroinflammatory cascade that typically accompanies BBB failure in these circumstances has been strongly linked to elevated levels of pro-inflammatory cytokines such as tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6). This mini review will examine our current knowledge of how cytokines may dysregulate the interendothelial paracellular pathway leading to elevated BBB permeability. The mechanistic role of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase)-induced oxidative stress in these events will also be addressed.

We have investigated the role of Notch receptor signaling in controlling human vascular smooth muscle cell (hVSMC) differentiation in vitro and established a role for cyclic strain-induced changes in Notch signaling in promoting this phenotypic response. The expression of ␣-actin, calponin, myosin, and smoothelin was examined by performing immunocytochemistry, Western blot analysis, and quantitative real-time PCR in hVSMCs cultured under static conditions after forced overexpression of constitutively active Notch 1 and 3 receptors, inhibition of endogenous Cp-binding factor 1 (CBF-1)/recombination signal sequence-binding protein-J (RBP-J) signaling, and exposure to cyclic strain using a Flexercell Tension Plus unit. Overexpression of constitutively active Notch intracellular (IC) receptors (Notch 1 IC and Notch 3 IC) resulted in a significant downregulation of ␣-actin, calponin, myosin, and smoothelin expression, an effect that was significantly attenuated after inhibition of Notch-mediated, CBF-1/RBP-J-dependent signaling by coexpression of RPMS-1 (EpsteinBarr virus-encoded gene product) and selective knockdown of basic helix-loop-helix factors [hairy enhancer of split (HES) gene and Hes-related transcription (Hrt) factors Hrt-1, Hrt-2, and Hrt-3] using targeted small interfering RNA. Cells cultured under conditions of defined equibiaxial cyclic strain (10% strain, 60 cycles/min, 24 h) exhibited a significant reduction in Notch 1 IC and Notch 3 IC expression concomitant with a significant increase in VSMC differentiation marker expression. Moreover, this cyclic strain-induced increase was further enhanced after inhibition of CBF-1/RBP-Jdependent signaling with RPMS-1. These findings suggest that Notch promotes changes in hVSMC phenotype via activation of CBF-1/ RBP-J-dependent pathways in vitro and contributes to the phenotypic response of VSMCs to cyclic strain-induced changes in VSMC differentiation.basic helix-loop-helix; cyclic strain; myosin; smoothelin MODIFICATIONS IN THE STRUCTURE, integrity, and function of arterial blood vessels are central to the pathogenesis of many vascular diseases (20). Numerous studies have demonstrated a distinct heterogeneity of vascular smooth muscle cell (VSMC) phenotype in the vessel walls of both human and animal models and in cell culture studies (6). Because adult VSMCs are not terminally differentiated, they are capable of changing phenotype in response to changes in local environmental cues, including growth factors and/or inhibitors, mechanical influences, cell-cell and cell-matrix interactions, and various inflammatory mediators (17, 18). The overall control of VSMC differentiation and the regulation of its responses to changing environmental cues is extremely complex and involves the cooperative interaction of many factors and signaling pathways that are only beginning to be understood (17,18).Studies in cultured VSMCs have implicated a large number of factors in regulating VSMC differentiation, including mechanical forces (5, 17), contractile agonists, extracellula...

Blood-brain barrier (BBB) regulation involves the coordinated interaction of intercellular adherens and tight junctions in response to stimuli. One such stimulus, shear stress, has been shown to upregulate brain microvascular endothelial cell (BMvEC) barrier function, although our knowledge of the signaling mechanisms involved is limited. In this article, we examined the hypothesis that VE-cadherin can transmit shear signals to tight junction occludin with consequences for pTyr-occludin and barrier function. In initial studies, chronic shear enhanced membrane localization of ZO-1 and claudin-5, decreased pTyr-occludin (in part via a dephostatin-sensitive mechanism), and reduced BMvEC permeability, with flow reduction in pre-sheared BMvECs having converse effects. In further studies, VE-cadherin inhibition (VE-cad ΔEXD) blocked shear-induced Rac1 activation, pTyr-occludin reduction, and barrier upregulation, consistent with an upstream role for VE-cadherin in transmitting shear signals to tight junctions through Rac1. As VE-cadherin is known to mediate Rac1 activation via Tiam1 recruitment, we subsequently confirmed that Tiam1 inhibition (Tiam1-C580) could elicit effects similar to VE-cad ΔEXD. Finally, the observed attenuation of shear-induced changes in pTyr-occludin level and barrier phenotype following Rac1 inhibition (NSC23766, T17N) establishes a downstream role for Rac1 in this pathway. In summary, we describe for the first time in BMvECs a role for VE-cadherin in the transmission of physiological shear signals to tight junction occludin through engagement of Tiam1/Rac1 leading to barrier stabilization. A downstream role is also strongly indicated for a protein tyrosine phosphatase in pTyr-occludin modulation. Importantly, these findings suggest an important route of inter-junctional signaling cross-talk during BBB response to flow.