Claudin-5 Controls Intercellular Barriers of Human Dermal Microvascular but Not Human Umbilical Vein Endothelial Cells

Article · January 2013with22 Reads
DOI: 10.1161/ATVBAHA.112.300893 · Source: PubMed
Objective: To assess the role claudin-5, an endothelial cell (EC) tight junction protein, plays in establishing basal permeability levels in humans by comparing claudin-5 expression levels in situ and analyzing junctional organization and function in 2 widely used models of cultured ECs, namely human dermal microvascular (HDM)ECs and human umbilical vein (HUV)ECs. Methods and results: By immunofluorescence microscopy, ECs more highly express claudin-5 (but equivalently express vascular endothelial-cadherin) in human dermal capillaries versus postcapillary venules and in umbilical and coronary arteries versus veins, correlating with known segmental differences in tight junction frequencies and permeability barriers. Postconfluent cultured HDMECs express more claudin-5 (but equivalent vascular endothelial-cadherin) and show higher transendothelial electric resistance and lower macromolecular flux than similarly cultured HUVECs. HDMEC junctions are more complex by transmission electron microscopy and show more continuous claudin-5 immunofluorescence than HUVEC junctions. Calcium chelation or dominant negative vascular endothelial-cadherin overexpression decreases transendothelial electric resistance and disrupts junctions in HUVECs, but not in HDMECs. Claudin-5 overexpression in HUVECs fails to increase transendothelial electric resistance or claudin-5 continuity, whereas claudin-5 knockdown in HDMECs, but not in HUVECs, reduces transendothelial electric resistance and increases antibody accessibility to junctional proteins. Conclusions: Claudin-5 expression and junctional organization control HDMEC and arteriolar-capillary paracellular barriers, whereas HUVEC and venular junctions use vascular endothelial-cadherin.
    • Both the size and the charge barriers of blood vessels are largely affected by the physiological state of the surrounding tissue interstitium. These changes in permeability were conventionally assessed by an in vitro transwell assay system that measured the flux of variable molecules through the endothelial cell monolayer cultured in transwell chambers under various stimulus agents[26][27][28][29]. Despite the utility of the assay, it has frequently been pointed out that this assay system might not reconstitute the actual vascular integrity and permeability in vivo (discussed in[27]).
    [Show abstract] [Hide abstract] ABSTRACT: Regulation of blood vessel permeability is essential for the homeostasis of peripheral tissues. This regulation controls the trafficking of plasma contents, including water, vitamins, ions, hormones, cytokines, amyloids, lipoproteins, carrier proteins, and immunoglobulins. The properties of blood vessels vary among tissues based on their structural differences: continuous, fenestrated, or sinusoidal. These three types of blood vessels have different charge and size barrier properties. The anionic luminal glycocalyx layer on endothelial cells establishes the “charge barrier” that repels the attachment of negatively charged blood cells and plasma molecules. In contrast, the “size barrier” of blood vessels largely relies on the interendothelial junctions (IEJs) between endothelial cells, which define the paracellular permeability. As in most peripheral tissues, blood capillaries in the skin are composed of continuous and/or fenestrated blood vessels that have relatively tighter IEJs compared to those in the internal organs. Small vesicles in the capillary endothelium were discovered in the 1950s, and studies have since confirmed that blood endothelial cells transport the plasma contents by endocytosis and subsequent transcytosis and exocytosis—this process is called transcellular permeability. The permeability of blood vessels is highly variable as a result of intrinsic and extrinsic factors. It is significantly elevated upon tissue inflammations as a result of disabled IEJs and increased paracellular permeability due to inflammatory mediators. An increase in transcellular permeability during inflammation has also been postulated. Here, we provide an overview of the general properties of vascular permeability based on our recent observations of murine skin inflammation models, and we discuss its physiological significance in peripheral homeostasis.
    Article · Dec 2017
    • Two SFK members, SRC and YES1, are tyrosine phosphorylated to transduce signals important for VEGF165-induced vascular permeability (Eliceiri et al., 1999; Scheppke et al., 2008). We therefore examined the requirement of NRP1 for VEGF165-induced SFK activation in human dermal microvascular ECs (HDM ECs), which are known to form monolayers with relatively well-organized intercellular contacts (Kluger et al., 2013 ). Immunostaining of confluent, nonpermeabilized HDM ECs in normal growth conditions showed NRP1 localization on the cell surface, including areas of cell– cell contact (Fig. 4 A).
    [Show abstract] [Hide abstract] ABSTRACT: The vascular endothelial growth factor (VEGF) isoform VEGF165 stimulates vascular growth and hyperpermeability. Whereas blood vessel growth is essential to sustain organ health, chronic hyperpermeability causes damaging tissue edema. By combining in vivo and tissue culture models, we show here that VEGF165-induced vascular leakage requires both VEGFR2 and NRP1, including the VEGF164-binding site of NRP1 and the NRP1 cytoplasmic domain (NCD), but not the known NCD interactor GIPC1. In the VEGF165-bound receptor complex, the NCD promotes ABL kinase activation, which in turn is required to activate VEGFR2-recruited SRC family kinases (SFKs). These results elucidate the receptor complex and signaling hierarchy of downstream kinases that transduce the permeability response to VEGF165. In a mouse model with choroidal neovascularisation akin to age-related macular degeneration, NCD loss attenuated vessel leakage without affecting neovascularisation. These findings raise the possibility that targeting NRP1 or its NCD interactors may be a useful therapeutic strategy in neovascular disease to reduce VEGF165-induced edema without compromising vessel growth.
    Full-text · Article · Mar 2017
    • The main barrier to transdermal drug delivery is the stratum corneum, outer layer of the epidermis filled with intercellular lipid matrix containing ceramide and non-living corneocyte cells with a dense keratin network. Further, the stratum corneum cells are connected by the tight junctions closely (Kluger et al., 2013). The stratum corneum barrier prevents not just the entry of foreign material and pathogens, but also moisture evaporation (Barry, 1991; Iwai et al., 2012 Iwai et al., , 2013 Obata et al., 2010 ).
    [Show abstract] [Hide abstract] ABSTRACT: In spite of numerous advantages, transdermal drug delivery systems are unfeasible for most drugs because of the barrier effect of the stratum corneum. Ionic liquids were recently used to enhance transdermal drug delivery by improving drug solubility. In the present study, safe and effective ionic liquids for transdermal absorption were obtained as salts generated by a neutralization reaction between highly biocompatible aliphatic carboxylic acids (octanoic acid or isostearic acid) and aliphatic amines (diisopropanolamine or triisopropanolamine) (Medrx Co., Ltd., 2009). The mechanism of skin permeability enhancement by ionic liquids were investigated by hydrophilic phenol red and hydrophobic turobuterol. Further, the skin permeation enhancing effect was remarkably superior in the acid excess state rather than the neutralization state. Infrared absorption spectrum analysis confirmed that ionic liquids/aliphatic carboxylic acid/aliphatic amine are coexisting at all mixing states. In the acid excess state, ionic liquids interact with aliphatic carboxylic acids via hydrogen bonds. Thus, the skin permeation enhancing effect is not caused by the ionic liquid alone. The “liquid salt mixture,” referred to as a complex of ingredients coexisting with ionic liquids, form a molecular assembly incorporating hydrophilic drug. This molecular assembly was considered an effective and safety enhancer of transdermal drug permeation.
    Article · Mar 2016
    • Junctions are dynamic structures whose regulation and structural changes strongly impact adhesion strength and tissue plasticity. ECs from different types of vessels and also from different organs show differences in junction composition and organization (Orsenigo et al., 2012; Kluger et al., 2013). Recent studies revealed that the cotranscriptional regulator YAP (Yes-associated protein), originally characterized as the molecular target of the size-controlling Hippo pathway (Varelas, 2014), is a key relay for the transmission of mechanical inputs into gene transcriptional programs (Dupont et al., 2011).
    [Show abstract] [Hide abstract] ABSTRACT: Vascular endothelial (VE)-cadherin transfers intracellular signals contributing to vascular hemostasis. Signaling through VE-cadherin requires association and activity of different intracellular partners. Yes-associated protein (YAP)/TAZ transcriptional cofactors are important regulators of cell growth and organ size. We show that EPS8, a signaling adapter regulating actin dynamics, is a novel partner of VE-cadherin and is able to modulate YAP activity. By biochemical and imaging approaches, we demonstrate that EPS8 associates with the VE-cadherin complex of remodeling junctions promoting YAP translocation to the nucleus and transcriptional activation. Conversely, in stabilized junctions, 14-3-3-YAP associates with the VE-cadherin complex, whereas Eps8 is excluded. Junctional association of YAP inhibits nuclear translocation and inactivates its transcriptional activity both in vitro and in vivo in Eps8-null mice. The absence of Eps8 also increases vascular permeability in vivo, but did not induce other major vascular defects. Collectively, we identified novel components of the adherens junction complex, and we introduce a novel molecular mechanism through which the VE-cadherin complex controls YAP transcriptional activity.
    Full-text · Article · Dec 2015
    • Although Claudin-5 is largely considered to be an endothelial specific tight junction member (Morita et al., 1999), it is also expressed in other cell types (Turksen and Troy, 2004). Claudin-5 up-regulation has been shown to enhance the blood–brain barrier (Honda et al., 2006), reduce paracellular cation permeability (Wen et al., 2004), and improve endothelial barrier function (Kluger et al., 2013). Furthermore, Claudin-5 knockout mice die within 1 day after birth with a compromised blood-brain barrier (Nitta et al., 2003).
    [Show abstract] [Hide abstract] ABSTRACT: Shear stress secondary to increased pulmonary blood flow (PBF) is elevated in some children born with congenital cardiac abnormalities. However, the majority of these patients do not develop pulmonary edema, despite high levels of permeability inducing factors. Previous studies have suggested that laminar fluid shear stress can enhance pulmonary vascular barrier integrity. However, little is known about the mechanisms by which this occurs. Using microarray analysis, we have previously shown that Sox18, a transcription factor involved in blood vessel development and endothelial barrier integrity, is up-regulated in an ovine model of congenital heart disease with increased PBF (shunt). By subjecting ovine pulmonary arterial endothelial cells (PAEC) to laminar flow (20 dyn/cm2), we identified an increase in trans-endothelial resistance (TER) across the PAEC monolayer that correlated with an increase in Sox18 expression. Further, the TER was also enhanced when Sox18 was over-expressed and attenuated when Sox18 expression was reduced, suggesting that Sox18 maintains the endothelial barrier integrity in response to shear stress. Further, we found that shear stress up-regulates the cellular tight junction protein, Claudin-5, in a Sox18 dependent manner, and Claudin-5 depletion abolished the Sox18 mediated increase in TER in response to shear stress. Finally, utilizing peripheral lung tissue of 4 week old shunt lambs with increased PBF, we found that both Sox18 and Claudin-5 mRNA and protein levels were elevated. In conclusion, these novel findings suggest that increased laminar flow protects endothelial barrier function via Sox18 dependent up-regulation of Claudin-5 expression.
    Article · Nov 2014
    • Plakoglobin in epithelial cells is mostly located at desmosomes, but in endothelial cells, which are devoid of desmosomes, it links VE-cadherin and acts in a way that is similar to b-catenin by promoting the anchorage to actin microfilaments (Bazzoni and Dejana, 2004; Vestweber et al., 2009; Komarova and Malik, 2010). Endothelial cells from different types of vessels can also show differences in junction composition and organization (Orsenigo et al., 2012; Kluger et al., 2013). The association of AJs to actin is necessary for junction assembly and maintenance.
    [Show abstract] [Hide abstract] ABSTRACT: Adherens junctions have an important role in the control of vascular permeability. These structures are located at cell-to-cell contacts, mediate cell adhesion and transfer intracellular signals. Adhesion is mediated by cadherins, which interact homophilically in trans and form lateral interactions in cis. VE-cadherin (also known as CDH5 and CD144) is the major component of endothelial adherens junctions and is specific to endothelial cells. Endothelial cells from different types of vessels, such as lymphatic vessels, arteries and veins, show differences in junction composition and organization. Vascular permeability is increased by modifications in the expression and function of adherens junction components. In some cases these defects might be cause of pathology. In this Cell Science at a Glance article, we present the example of the so-called cerebral cavernous malformation (CCM), where adherens junctions are dismantled in the vessels contributing to brain microcirculation. This causes the loss of endothelial cell apical-basal polarity and the formation of cavernomas, which are fragile and hemorrhagic. Other diseases are accompanied by persistent alterations of vascular morphology and permeability, such as seen in tumors. It will be important to achieve a better understanding of the relationship between vascular fragility, malformations and junctional integrity in order to develop more effective therapies.
    Article · Jun 2013
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