Plasminogen Activator Inhibitor-1 Controls Vascular Integrity by Regulating VE-Cadherin Trafficking

Background

Plasminogen activator inhibitor-1 (PAI-1), a serine protease inhibitor, is expressed and secreted by endothelial cells. Patients with PAI-1 deficiency show a mild to moderate bleeding diathesis, which has been exclusively ascribed to the function of PAI-1 in down-regulating fibrinolysis. We tested the hypothesis that PAI-1 function plays a direct role in controlling vascular integrity and permeability by keeping endothelial cell-cell junctions intact.

Methodology/Principal Findings

We utilized PAI-039, a specific small molecule inhibitor of PAI-1, to investigate the role of PAI-1 in protecting endothelial integrity. In vivo inhibition of PAI-1 resulted in vascular leakage from intersegmental vessels and in the hindbrain of zebrafish embryos. In addition PAI-1 inhibition in human umbilical vein endothelial cell (HUVEC) monolayers leads to a marked decrease of transendothelial resistance and disrupted endothelial junctions. The total level of the endothelial junction regulator VE-cadherin was reduced, whereas surface VE-cadherin expression was unaltered. Moreover, PAI-1 inhibition reduced the shedding of VE-cadherin. Finally, we detected an accumulation of VE-cadherin at the Golgi apparatus.

Conclusions/Significance

Our findings indicate that PAI-1 function is important for the maintenance of endothelial monolayer and vascular integrity by controlling VE-cadherin trafficking to and from the plasma membrane. Our data further suggest that therapies using PAI-1 antagonists like PAI-039 ought to be used with caution to avoid disruption of the vessel wall.     

Binding to the Extracellular Matrix and Proteolytic Processing: Two Key Mechanisms Regulating Vascular Endothelial Growth Factor Action

Vascular endothelial growth factor (VEGF, VEGF-A) is a major regulator of physiological and pathological angiogenesis. One feature of VEGF is the existence of multiple isoforms arising from alternative exon splicing. Our initial biochemical and biological studies indicated that such isoforms are uniquely suited to generate angiogenic gradients by virtue of their differential ability to interact with the extracellular matrix (ECM). Although ECM-bound VEGF was bioactive, processing by physiologically relevant proteases such as plasmin was identified as a key mechanism to convert ECM-bound VEGF into freely diffusible forms. This retrospective article examines the early studies and also emphasizes the subsequent progress in our understanding of these processes in health and disease.     

Signaling by the Extracellular Matrix Protein Reelin Promotes Granulosa Cell Proliferation in the Chicken Follicle

Chicken oocytes develop in follicles and reach an enormous size because of a massive uptake of yolk precursors such as very low density lipoprotein and vitellogenin. Oocyte growth is supported by theca cells and granulosa cells, which establish dynamic and highly organized cell layers surrounding the oocyte. The signaling processes orchestrating the development of these layered structures are largely unknown. Here we demonstrate that the Reelin pathway, which determines the development of layered neuronal structures in the brain, is also active in chicken follicles. Reelin, which is expressed in theca cells, triggers a signal in granulosa cells via apolipoprotein E receptor 2 and the very low density lipoprotein receptor, resulting in the phosphorylation of disabled-1 and consecutive activation of the phosphatidylinositol 3-kinase/Akt pathway. This signaling pathway supports the proliferation of differentiated granulosa cells to keep up with the demand of cells to cover the rapidly increasing surface of the giant germ cell.     

An O-Glycosyltransferase Promotes Cell Adhesion during Development by Influencing Secretion of an Extracellular Matrix Integrin Ligand

Protein secretion and localization are crucial during eukaryotic development, establishing local cell environments as well as mediating cell interactions, signaling, and adhesion. In this study, we demonstrate that the glycosyltransferase, pgant3, specifically modulates integrin-mediated cell adhesion by influencing the secretion and localization of the integrin ligand, Tiggrin. We demonstrate that Tiggrin is normally O-glycosylated and localized to the basal matrix where the dorsal and ventral cell layers adhere in wild type Drosophila wings. In pgant3 mutants, Tiggrin is no longer O-glycosylated and fails to be properly secreted to this basal cell layer interface, resulting in disruption of integrin-mediated cell adhesion in the wing. pgant3-mediated effects are dependent on enzymatic activity, as mutations that form a stable protein yet abrogate O-glycosyltransferase activity result in Tiggrin accumulation within the dorsal and ventral cells comprising the wing. Our results provide the first in vivo evidence for the role of O-glycosylation in the secretion of specific extracellular matrix proteins, thus altering the composition of the cellular “microenvironment” and thereby modulating developmentally regulated cell adhesion events. As alterations in cell adhesion are a hallmark of cancer progression, this work provides insight into the long-standing association between aberrant O-glycosylation and tumorigenesis.     

The Deubiquitinating Enzyme USP8 Promotes Trafficking and Degradation of the Chemokine Receptor 4 at the Sorting Endosome

Reversible ubiquitination orchestrated by the opposition of ubiquitin ligases and deubiquitinating enzymes mediates endocytic trafficking of cell surface receptors for lysosomal degradation. Ubiquitin-specific protease 8 (USP8) has previously been implicated in endocytosis of several receptors by virtue of their deubiquitination. The present study explores an indirect role for USP8 in cargo trafficking through its regulation of the chemokine receptor 4 (CXCR4). Contrary to the effects of USP8 loss on enhanced green fluorescent protein, we find that USP8 depletion stabilizes CXCR4 on the cell surface and attenuates receptor degradation without affecting its ubiquitination status. In the presence of ligand, diminished CXCR4 turnover is accompanied by receptor accumulation on enlarged early endosomes and leads to enhancement of phospho-ERK signaling. Perturbation in CXCR4 trafficking, resulting from USP8 inactivation, occurs at the ESCRT-0 checkpoint, and catalytic mutation of USP8 specifically targeted to the ESCRT-0 complex impairs the spatial and temporal organization of the sorting endosome. USP8 functionally opposes the ubiquitin ligase AIP4 with respect to ESCRT-0 ubiquitination, thereby promoting trafficking of CXCR4. Collectively, our findings demonstrate a functional cooperation between USP8, AIP4, and the ESCRT-0 machinery at the early sorting phase of CXCR4 and underscore the versatility of USP8 in shaping trafficking events at the early-to-late endosome transition.     

Secretion of Acid Phosphatase by x4xenic Entamoeba histolytica NIH-200 and Properties of the Extracellular Enzyme

Entamoeba histolytica (NIH-200) secreted large amounts of acid phosphatase in its external environment when grown axenically in modified TPS-II medium. Fractionation by DEAE-cellulose chromatography of the precipitate obtained from the cell-free medium at 60% ammonium sulfate saturation yielded 3 distinct peaks of enzyme activity. The enzyme in all the peaks showed resistance to tartrate but was inhibited by fluoride, cupnc chloride, ethylene diamine-tetra acetic acid, ammonium molybdale and cysteine: however, enzyme associated with different peaks differed in its polyacrylamide gel electrophoretic profiles and behavior towards concanavalin A.     

The Mup-4 Locus in Caenorhabditis Elegans Is Essential for Hypodermal Integrity, Organismal Morphogenesis and Embryonic Body Wall Muscle Position

mup-4 is a member of a set of genes essential for correct embryonic body wall muscle cell positions in Caenorhabditis elegans. The mup-4 phenotype is variably expressed and three discrete arrest phenotypes arise during the phase of embryonic development when the worm elongates from a ball of cells to its worm shape (organismal morphogenesis). Mutants representing two of the phenotypic classes arrest without successful completion of elongation. Mutants of the third phenotypic class arrest after completion of elongation. Mutants that arrest after elongation display profound dorsal and ventral body wall muscle cell position abnormalities and a characteristic kinked body shape (the Mup phenotype) due to the muscle cell position abnormalities. Significantly, genetic mosaic analysis of mup-4 mutants demonstrates that mup-4 gene function is essential in the AB lineage, which generates most of the hypodermis (epidermis), a tissue with which muscle interacts. Consistent with the genetic mosaic data, phenotypic characterizations reveal that mutants have defects in hypodermal integrity and morphology. Our analyses support the conclusion that mup-4 is essential for hypodermal function and that this function is necessary for organismal morphogenesis and for the maintenance of body wall muscle position.     

Mechanical Stretch Suppresses microRNA-145 Expression by Activating Extracellular Signal-Regulated Kinase 1/2 and Upregulating Angiotensin-Converting Enzyme to Alter Vascular Smooth Muscle Cell Phenotype

Phenotype modulation of vascular smooth muscle cells (VSMCs) plays an important role in the pathogenesis of various vascular diseases, including hypertension and atherosclerosis. Several microRNAs (miRNAs) were found involved in regulating the VSMC phenotype with platelet-derived growth factor (PDGF) treatment, but the role of miRNAs in the mechanical stretch-altered VSMC phenotype is not clear. Here, we identified miR-145 as a major miRNA contributing to stretch-altered VSMC phenotype by miRNA array, quantitative RT-PCR and gain- and loss-of-function methods. Our data demonstrated that 16% stretch suppressed miR-145 expression, with reduced expression of contractile markers of VSMCs cultured on collagenI; overexpression of miR-145 could partially recover the expression in stretched cells. Serum response factor (SRF), myocardin, and Kruppel-like factor 4 (KLF4) are major regulators of the VSMC phenotype. The effect of stretch on myocardin and KLF4 protein expression was altered by miR-145 mimics, but SRF expression was not affected. In addition, stretch-activated extracellular signal-regulated kinase 1/2 (ERK1/2) and up-regulated angiotensin-converting enzyme (ACE) were confirmed to be responsible for the inhibition of miR-145 expression. Mechanical stretch inhibits miR-145 expression by activating the ERK1/2 signaling pathway and promoting ACE expression, thus modulating the VSMC phenotype.     

The RON Receptor Tyrosine Kinase Promotes Metastasis by Triggering MBD4-Dependent DNA Methylation Reprogramming

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The Neuropilin-1 Inhibitor,ATWLPPR Peptide,Prevents Experimental Diabetes-Induced Retinal Injury by Preserving Vascular Integrity and Decreasing Oxidative Stress

Neuropilin-1 (NRP-1) is a transmembrane glycoprotein. As a VEGF co-receptor, NRP1 significantly enhances VEGFR2 signaling and promotes vascular permeability and migration. The purpose of this study was to evaluate the effects of an NRP-1 inhibitor, ATWLPPR peptide, on the early stages of diabetic retinopathy. Eight-week-old male C57BL/6 mice were divided into three groups: a Normal group, a Diabetes (DB) ATWLPPR treatment group and a DB saline group. Electroretinography (ERG), fundus fluorescence angiography (FFA) and leukostasis were examined to evaluate the retinal injury induced by diabetes at the end of the fifth week after STZ injection. Occludin expression and extravasation of albumin were measured to determine the extent of vascular injury. The oxidative stress level and the levels of inflammation-associated proteins were also assayed. The results indicated that treatment with ATWLPPR prevents the abnormal condition of ERG (amplitudes of b-wave decreased and implicit time increased) and vascular injury (occludin degradation and increase in extravasated albumin). These effects were associated with a reduction in the oxidase stress level and the expression of VEGF, GFAP, and ICAM-1. We conclude that ATWLPPR, an NRP-1 inhibitor, may reduce the early retinal damage induced by diabetes by preserving vascular integrity and decreasing the oxidative stress level. Blockade of NRP-1 may be a new therapeutic strategy for the early stages of DR.     

The Interaction between Rice ERF3 and WOX11 Promotes Crown Root Development by Regulating Gene Expression Involved in Cytokinin Signaling

Crown roots are the main components of the fibrous root system in rice (Oryzasativa). WOX11, a WUSCHEL-relatedhomeobox gene specifically expressed in the emerging crown root meristem, is a keyregulator in crown root development. However, the nature of WOX11function in crown root development has remained elusive. Here, we identified a riceAP2/ERF protein, ERF3, which interacts with WOX11 and was expressed in crown rootinitials and during crown root growth. Functional analysis revealed thatERF3 was essential for crown root development and acts in auxin-and cytokinin-responsive gene expression. Downregulation of ERF3 inwox11 mutants produced a more severe root phenotype. Also,increased expression of ERF3 could partially complementwox11, indicating that the two genes functioned cooperatively toregulate crown root development. ERF3 and WOX11shared a common target, the cytokinin-responsive gene RR2. Theexpression of ERF3 and WOX11 only partiallyoverlapped, underlining a spatio-temporal control of RR2 expressionand crown root development. Furthermore, ERF3-regulated RR2expression was involved in crown root initiation, while the ERF3/WOX11 interactionlikely repressed RR2 during crown root elongation. These resultsdefine a mechanism regulating gene expression involved in cytokinin signaling duringdifferent stages of crown root development in rice.     

The Effects of Age and the Expression of SPARC on Extracellular Matrix Production by Cardiac Fibroblasts in 3-D Cultures

Fibrillar collagen is the primary component of the cardiac interstitial extracellular matrix. This extracellular matrix undergoes dramatic changes from birth to adulthood and then into advanced age. As evidence, fibrillar collagen content was compared in sections from neonates, adult, and old hearts and was found to increase at each respective age. Cardiac fibroblasts are the principle cell type that produce and control fibrillar collagen content. To determine whether fibroblast production, processing, and deposition of collagen differed with age, primary cardiac fibroblasts from neonate, adult, and old mice were isolated and cultured in 3-dimensional (3D) fibrin gels. Fibroblasts from each age aligned in fibrin gels along points of tension and deposited extracellular matrix. By confocal microscopy, wild-type neonate fibroblasts appeared to deposit less collagen into fibrillar structures than fibroblasts from adults. However, by immunoblot analysis, differences in procollagen production and processing of collagen I were not detected in neonate versus adult fibroblasts. In contrast, fibroblasts from old mice demonstrated increased efficiency of procollagen processing coupled with decreased production of total collagen. SPARC is a collagen-binding protein previously shown to affect cardiac collagen deposition. Accordingly, in the absence of SPARC, less collagen appeared to be associated with fibroblasts of each age grown in fibrin gels. In addition, the increased efficiency of procollagen alpha 1(I) processing in old wild-type fibroblasts was not detected in old SPARC-null fibroblasts. Increased levels of fibronectin were detected in wild-type neonate fibroblasts over that of adult and old fibroblasts but not in SPARC-null neonate fibroblasts versus older ages. Immunostaining of SPARC overlapped with that of collagen I but not to that of fibronectin in 3D cultures. Hence, whereas increases in procollagen processing, influenced by SPARC expression, plausibly contribute to increased collagen deposition in old hearts, other cellular mechanisms likely affect differential collagen deposition by neonate fibroblasts.