Characterization of cell-cell junction changes associated with the formation of a strong endothelial barrier

McRae, MaryPeace; LaFratta, Lindsay M.; Nguyen, Benjamin; Paris, Jason J.; Hauser, Kurt F.; Conway, Daniel E.

doi:10.1080/21688370.2017.1405774

Cited by 26 publications

(18 citation statements)

References 35 publications

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“…This was not surprising given the significant evidence that these supplements improve the barrier phenotype in ECs [24,25]. Of specific relevance, one study reported increased TEER and localization of ZO-1 and VE-cadherin, showing a more linear morphology (assessed qualitatively) in HUVECs treated with the same concentrations of cAMP supplements for 1 day, supporting our results in this study [31]. Interestingly, increasing culture time in the presence of cAMP did not increase junction coverage for ZO-1 or VE-cadherin, and required longer cAMP treatment to reach similar presentation values observed in shorter experiments (FBN results presented in Additional file 1: Figure S16).…”

Section: Discussionsupporting

confidence: 89%

“…Samples were treated with medium containing cAMP supplements: 250 μM 8-CPT-cAMP (Abcam, ab120424) and 17.5 μM RO-20-1724 (Tocris Bioscience, 0415), for 1, 3, or 6 days, or control HBMEC medium. These supplements are routinely used in EC culture to improve junction localization and barrier properties [4,[30][31][32][33]. For all experiments, the medium was first changed the day after cell seeding, then again on Days 3, 4, and 6 for the respective culture lengths.…”

Section: Substrate Coating and Experimental Conditionsmentioning

confidence: 99%

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Quantitatively relating brain endothelial cell–cell junction phenotype to global and local barrier properties under varied culture conditions via the Junction Analyzer Program

Gray

Jung

Inglut

et al. 2020

Fluids Barriers CNS

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Background: The endothelial cell-cell junctions of the blood-brain barrier (BBB) play a pivotal role in the barrier's function. Altered cell-cell junctions can lead to barrier dysfunction and have been implicated in several diseases. Despite this, the driving forces regulating junctional protein presentation remain relatively understudied, largely due to the lack of efficient techniques to quantify their presentation at sites of cell-cell adhesion. Here, we used our novel Junction Analyzer Program (JAnaP) to quantify junction phenotype (i.e., continuous, punctate, or perpendicular) in response to various substrate compositions, cell culture times, and cAMP treatments in human brain microvascular endothelial cells (HBMECs). We then quantitatively correlated junction presentation with barrier permeability on both a "global" and "local" scale. Methods:We cultured HBMECs on collagen I, fibronectin, collagen IV, laminin, fibronectin/collagen IV/laminin, or hyaluronic acid/gelatin for 2, 4, and 7 days with varying cAMP treatment schedules. Images of immunostained ZO-1, VE-cadherin, and claudin-5 were analyzed using the JAnaP to calculate the percent of the cell perimeter presenting continuous, punctate, or perpendicular junctions. Transwell permeability assays and resistance measurements were used to measure bulk ("global") barrier properties, and a "local" permeability assay was used to correlate junction presentation proximal to permeable monolayer regions.Results: Substrate composition was found to play little role in junction presentation, while cAMP supplements significantly increased the continuous junction architecture. Increased culture time required increased cAMP treatment time to reach similar ZO-1 and VE-cadherin coverage observed with shorter culture, though longer cultures were required for claudin-5 presentation. Prolonged cAMP treatment (6 days) disrupted junction integrity for all three junction proteins. Transwell permeability and TEER assays showed no correlation with junction phenotype, but a local permeability assay revealed a correlation between the number of discontinuous and no junction regions with barrier penetration. Conclusions:These results suggest that cAMP signaling influences HBMEC junction architecture more than matrix composition. Our studies emphasized the need for local barrier measurement to mechanistically understand the role of junction phenotype and supported previous results that continuous junctions are indicative of a more mature/ © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article' s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material...

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Section: Discussionsupporting

confidence: 89%

Section: Substrate Coating and Experimental Conditionsmentioning

confidence: 99%

Quantitatively relating brain endothelial cell–cell junction phenotype to global and local barrier properties under varied culture conditions via the Junction Analyzer Program

Gray

Jung

Inglut

et al. 2020

Fluids Barriers CNS

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“…Many studies have shown that poloxamers are capable of sealing the compromised cell membrane. For example, the FDA-approved poloxamers P188 was demonstrated to reconstitute the membrane in BBB 30,31 and down-regulated the secretion of matrix metalloproteinases (MMP) 32,33 by likely modulating the TNF-α pathway 34 . In this study, we cultured a monolayer of brain endothelial cells on a well-characterized synthetic membrane and quantitatively determined changes in the permeability and disorganized tight junctions in response to the blast-induced microcavitation.…”

mentioning

confidence: 99%

Modulation of in vitro Brain Endothelium by Mechanical Trauma: Structural and Functional Restoration by Poloxamer 188

Inyang

Abhyankar

Chen

et al. 2020

Sci Rep

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Brain injuries caused by an explosive blast or blunt force is typically presumed to associate with mechanical trauma to the brain tissue. Recent findings from our laboratory suggest that shockwaves produced by a blast can generate micron-sized bubbles in the tissue. the collapse of microbubbles (i.e., microcavitation) may induce a mechanical trauma and compromise the integrity of the blood-brain endothelium (BBE). To test our hypothesis, we engineered a BBE model to determine the effect of microbubbles on the structural and functional changes in the BBe. Using monolayers of mouse primary brain microvascular endothelial cells, the permeability coefficient was measured following simulated blast-induced microcavitation. this event down-regulated the expression of tight junction markers, disorganized the cell-cell junction, and increased permeability. Since poloxamers have been shown to rescue damaged cells, the cells were treated with the FDA-approved poloxamer 188 (P188). The results indicate P188 recovered the permeability, restored the tight junctions, and suppressed the expressions of matrix metalloproteinases. the biomimetic interface we developed appears to provide a systematic approach to replicate the structure and function of BBe, determine its alteration in response to traumatic brain injury, and test potential therapeutic treatments to repair the damaged brain endothelium. Traumatic brain injury (TBI) is one of the major causes of emergency visits and hospitalization. In 2010, the Centers for Disease Control reported about 2.5 million emergency department visits, hospitalizations, and deaths here in the United States alone 1. TBIs are also caused by an explosive blast or blunt force to the head especially among those who serve in the U.S. military 2-4. The trauma can lead to endothelial cell detachment, tight junction disruption, and altered blood-brain barrier (BBB) permeability 5,6. One of the unique features of BBB is the regulation of biotransport through a monolayer of brain endothelial cells (BECs). BECs are highly regulated in their structure and function by the tight junction complex that is composed of, among many molecules, zonula occludens (ZO-1) and the occludins family 7,8. Only particles with a molecular mass of less than 500 Daltons can cross the BBB efficiently 9. However, the structural integrity of the BBB can be mechanically and biochemically compromised 10-13 , allowing harmful substances to extravasate into the brain. The leaky brain endothelium, in turn, may lead to secondary brain injury 14-16. Several sophisticated TBI models of explosive blast or blunt force have been studied. However, the potential mechanisms connecting shock wave exposure to the head and to TBI are still not well understood 17. One of the mechanical traumas that have been investigated in our laboratory is the formation of micron-size bubbles in response to shock wave and subsequent collapse of such microbubbles, referred to as microcavitation 18. The collapse of highly pressurized microbubbles is thought to prod...

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“…Oxidative stress is a primary mediator of neurologic injury following ischemic stroke [ 49 ] and entails various mechanisms of the “ischemic cascade,” which leads to cell death and indicates that oxidative stress may become a potential therapeutic target of ischemic stroke. Cell junctions may play key roles in regulation of BBB stability and permeability to paracellular compounds [ 50 ]. It was thought that several QKL targets are expressed in cell junctions indicating a BBB modulating effect.…”

Section: Discussionmentioning

confidence: 99%

Network Pharmacology‐Based Approach to Revealing Biological Mechanisms of Qingkailing Injection against IschemicStroke: Focusing on Blood‐Brain Barrier

Zhang

Wang

Cheng

et al. 2020

Evidence-Based Complementary and Alternative Medicine

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Ischemic stroke is the most common type of cerebrovascular accident worldwide. It causes long-term disability and death. Qingkailing (QKL) injection is a traditional Chinese patent medicine which has been clinically applied in the treatment of ischemic stroke for nearly thirty years. In the present study, network pharmacology combined with experimentation was used to elucidate the mechanisms of QKL. ADME screening and target prediction identified 62 active compounds and 275 targets for QKL. Topological screening of the protein-protein interaction (PPI) network was used to build a core PPI network consisting of 408 nodes and 17,830 edges. KEGG enrichment indicated that the main signaling pathway implicated in ischemic stroke involved hypoxia-inducible factor-1 (HIF-1). Experimentation showed that QKL alleviated neurological deficits, brain infraction, blood-brain barrier (BBB) leakage, and tight junction degeneration in a mouse ischemic stroke model. Two-photon laser scanning microscopy was used to evaluate BBB permeability and cerebral microvessel structure in living mice. HIF-1α, matrix metalloproteinase-9 (MMP-9), and tight junction proteins such as occludin, zonula occludins-1 (ZO-1), claudin-5, and VE-Cadherin were measured by western blotting. QKL upregulated ZO-1 and downregulated HIF-1α and MMP-9. QKL has a multiapproach, multitarget, and synergistic effect against ischemic stroke.

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Characterization of cell-cell junction changes associated with the formation of a strong endothelial barrier

Cited by 26 publications

References 35 publications

Quantitatively relating brain endothelial cell–cell junction phenotype to global and local barrier properties under varied culture conditions via the Junction Analyzer Program

Quantitatively relating brain endothelial cell–cell junction phenotype to global and local barrier properties under varied culture conditions via the Junction Analyzer Program

Modulation of in vitro Brain Endothelium by Mechanical Trauma: Structural and Functional Restoration by Poloxamer 188

Network Pharmacology‐Based Approach to Revealing Biological Mechanisms of Qingkailing Injection against IschemicStroke: Focusing on Blood‐Brain Barrier

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