Transendothelial Electrical Resistance Research Articles

Novel truncating variants in CTNNB1 cause familial exudative vitreoretinopathy

BackgroundFamilial exudative vitreoretinopathy (FEVR) is an inheritable blinding disorder with clinical and genetic heterogeneity. Heterozygous variants in the CTNNB1 gene have been reported to cause FEVR. However, the pathogenic basis...

Vascular inflammation on a chip: A scalable platform for trans-endothelial electrical resistance and immune cell migration.

The vasculature system plays a critical role in inflammation processes in the body. Vascular inflammatory mechanisms are characterized by disruption of blood vessel wall permeability together with increased immune cell recruitment and migration. There is a critical need to develop models that fully recapitulate changes in vascular barrier permeability in response to inflammatory conditions. We developed a scalable platform for parallel measurements of trans epithelial electrical resistance (TEER) in 64 perfused microfluidic HUVEC tubules under inflammatory conditions. Over 250 tubules where exposed to Tumor necrosis factor alpha (TNFα) and interferon gamma (INF-γ) or human peripheral blood mononuclear cells. The inflammatory response was quantified based on changes TEER and expression of ICAM and VE-cadherin. We observed changes in barrier function in the presence of both inflammatory cytokines and human peripheral blood mononuclear cells, characterized by decreased TEER values, increase in ICAM expression as well changes in endothelial morphology. OrganoPlate 3-lane64 based HUVEC tubules provide a valuable tool for inflammatory studies in an automation compatible manner. Continuous TEER measurements enable long term, sensitive assays for barrier studies. We propose the use of our platform as a powerful tool for modelling endothelial inflammation in combination with immune cell interaction that can be used to screen targets and drugs to treat chronic vascular inflammation.

Senescence in brain pericytes attenuates blood-brain barrier function in vitro: A comparison of serially passaged and isolated pericytes from aged rat brains

Aging is associated with the dysfunction of the blood-brain barrier (BBB), which comprises brain microvessel endothelial cells (BMECs), astrocytes, and pericytes. Pericytes are present at intervals along the walls of the brain capillaries and play a key role in maintaining BBB integrity. Accumulation of senescent cells and the senescence-associated secretory phenotype (SASP) in the brain facilitate the development of age-related neurodegenerative diseases with BBB dysfunction. However, the ability of pericytes to support BBB integrity and their correlation with cellular senescence or aging remain unknown. Here, we investigated cellular senescence in pericytes focusing on its impact on BBB function using BBB models comprising intact BMECs co-cultured with senescent pericytes, which were obtained through a serial passage or isolated from 18-month-old rats. To assess BBB function, transendothelial electrical resistance (TEER) and permeability of sodium fluorescein (Na–F) were studied. Both serially passaged pericytes (in passage 4, 7, and 10) and aged pericytes isolated from 18-month-old rats showed decreased TEER and enhanced permeability of BMECs to Na–F compared to that of normal pericytes (passage 2 or young). Furthermore, serially passaged and aged pericytes showed characteristic features of cellular senescence, including increased β-galactosidase activity, cell cycle arrest, enhanced expression of mRNA, and SASP factors. However, the senescence-induced mRNA expression profile of pericyte markers varied between serially passaged and aged pericytes. Hence, in vitro serial passages and isolation from naturally aged rodents differently influenced genetic and biochemical features of senescent brain pericytes. We conclude that senescent brain pericytes can induce BBB dysfunction and those isolated from aged rodents retain the senescence-specific properties. Our findings provide an alternative tool to investigate the senescence in brain pericytes in vitro.

Protective Effects of Human Pericyte-like Adipose-Derived Mesenchymal Stem Cells on Human Retinal Endothelial Cells in an In Vitro Model of Diabetic Retinopathy: Evidence for Autologous Cell Therapy.

Diabetic retinopathy (DR) is characterized by morphologic and metabolic alterations in endothelial cells (ECs) and pericytes (PCs) of the blood-retinal barrier (BRB). The loss of interendothelial junctions, increased vascular permeability, microaneurysms, and finally, EC detachment are the main features of DR. In this scenario, a pivotal role is played by the extensive loss of PCs. Based on previous results, the aim of this study was to assess possible beneficial effects exerted by adipose mesenchymal stem cells (ASCs) and their pericyte-like differentiated phenotype (P-ASCs) on human retinal endothelial cells (HRECs) in high glucose conditions (25 mM glucose, HG). P-ASCs were more able to preserve BRB integrity than ASCs in terms of (a) increased transendothelial electrical resistance (TEER); (b) increased expression of adherens junction and tight junction proteins (VE-cadherin and ZO-1); (c) reduction in mRNA levels of inflammatory cytokines TNF-α, IL-1β, and MMP-9; (d) reduction in the angiogenic factor VEGF and in fibrotic TGF-β1. Moreover, P-ASCs counteracted the HG-induced activation of the pro-inflammatory phospho-ERK1/2/phospho-cPLA2/COX-2 pathway. Finally, crosstalk between HRECs and ASCs or P-ASCs based on the PDGF-B/PDGFR-β axis at the mRNA level is described herein. Thus, P-ASCs might be considered valuable candidates for therapeutic approaches aimed at countering BRB disruption in DR.

Ultra-thin and ultra-porous nanofiber networks as a basement-membrane mimic.

Current basement membrane (BM) mimics used for modeling endothelial and epithelial barriers in vitro do not faithfully recapitulate key in vivo physiological properties such as BM thickness, porosity, stiffness, and fibrous composition. Here, we use networks of precisely arranged nanofibers to form ultra-thin (∼3 μm thick) and ultra-porous (∼90%) BM mimics for blood-brain barrier modeling. We show that these nanofiber networks enable close contact between endothelial monolayers and pericytes across the membrane, which are known to regulate barrier tightness. Cytoskeletal staining and transendothelial electrical resistance (TEER) measurements reveal barrier formation on nanofiber membranes integrated within microfluidic devices and transwell inserts. Further, significantly higher TEER values indicate a biological benefit for co-cultures formed on the ultra-thin nanofiber membranes. Our BM mimic overcomes critical technological challenges in forming co-cultures that are in proximity and facilitate cell-cell contact, while still being constrained to their respective sides. We anticipate that our nanofiber networks will find applications in drug discovery, cell migration, and barrier dysfunction studies.

Corneal Endothelial Cell Cultures from Organotypic Preservation of Older Donor Corneas Are Suitable for Advanced Cell Therapy

Introduction: The purpose of this work was to evaluate the in vitro growth capacity and functionality of human corneal endothelial cells (hCEC) expanded from corneas of elderly (>60 years) donors that were preserved using an organotypic culture method (>15 days, 31°C) and did not meet the clinical criteria for keratoplasty. Methods: Cell cultures were obtained from prior descemetorhexis (≥10 mm) and a controlled incubation with collagenase type I followed by recombinant trypsin. Cells were seeded on coated plates (fibronectin-albumin-collagen I) and cultures were expanded using the dual supplemented medium approach (maintenance medium and growth medium), in the presence of a 10 μm Rho-associated protein kinase inhibitor (Y-27632). Cell passages were obtained at culture confluency (∼2 weeks). A quantitative colorimetric WST-1 cell growth assay was performed at different time points of the culture. Morphometric analysis (area assessment and circularity), immunocytochemistry (ZO-1, Na+/K+-ATPase α, Ki67), and transendothelial electrical resistance (TEER) were performed on confluent monolayers. Results: There was no difference between the cell growth profiles of hCEC cultures obtained from corneas older than 60 years, whether preserved cold or cultivated organotypic corneas. Primary cultures were able to maintain a certain cell circularity index (around 0.8) and morphology (hexagonal) similar to corneal endothelial mosaic. The ZO-1 and Na+/K+-ATPase pump markers were highly positive in confluent cell monolayers at 21 days after isolation (passage 0; P0), but significantly decreased in confluent monolayers after the first passage (P1). A weak expression of Ki67 was observed in both P0 and P1 monolayers. The P0 monolayers showed a progressive increase in TEER values between days 6 and 11 and remained stable until day 18 of culture, indicating a state of controlled permeability in monolayers. The P1 monolayers also showed some functional ability but with decreased TEER values compared to monolayers at P0. Conclusions: Our results indicate that it is possible to obtain functional hCEC cultures in eye banks, using simplified and standardized protocols, from older donor corneas (>60 years of age), previously preserved under organotypic culture conditions. This tissue is more readily available in our setting, due to the profile of the donor population or due to the low endothelial count (<2,000 cells/mm2) of the donated cornea.

Perillyl alcohol modulates activation, permeability and integrity of human brain endothelial cells induced by Plasmodium falciparum.

Cerebral malaria (CM) is a severe immunovasculopathy caused for Plasmodium falciparum infection, which is characterised by the sequestration of parasitised red blood cells (pRBCs) in brain microvessels. Previous studies have shown that some terpenes, such as perillyl alcohol (POH), exhibit a marked efficacy in preventing cerebrovascular inflammation, breakdown of the brain-blood barrier (BBB) and brain leucocyte accumulation in experimental CM models. To analyse the effects of POH on the endothelium using human brain endothelial cell (HBEC) monolayers co-cultured with pRBCs. The loss of tight junction proteins (TJPs) and features of endothelial activation, such as ICAM-1 and VCAM-1 expression were evaluated by quantitative immunofluorescence. Microvesicle (MV) release by HBEC upon stimulation by P. falciparum was evaluated by flow cytometry. Finally, the capacity of POH to revert P. falciparum-induced HBEC monolayer permeability was examined by monitoring trans-endothelial electrical resistance (TEER). POH significantly prevented pRBCs-induced endothelial adhesion molecule (ICAM-1, VCAM-1) upregulation and MV release by HBEC, improved their trans-endothelial resistance, and restored their distribution of TJPs such as VE-cadherin, Occludin, and JAM-A. POH is a potent monoterpene that is efficient in preventing P. falciparum-pRBCs-induced changes in HBEC, namely their activation, increased permeability and alterations of integrity, all parameters of relevance to CM pathogenesis.

PLD2 deletion alleviates disruption of tight junctions in sepsis-induced ALI by regulating PA/STAT3 phosphorylation pathway.

Increased inflammatory exudation caused by endothelium and endothelial junction damage is a typical pathological feature of acute respiratory distress syndrome/acute lung injury (ARDS/ALI). Previous studies have shown that phospholipase D2 (PLD2) can increase the inflammatory response and has a close relationship with the severity of sepsis-induced ALI and the mortality of sepsis, but its mechanism is unknown. This study explored the effect and mechanism of PLD2 deletion on the structure and function of endothelial tight junction (TJ) in lipopolysaccharide (LPS)-induced ALI. We used C57BL/6 mice (wild-type and PLD2 knockout (PLD2-/-)) and human umbilical vein endothelial cell (HUVEC) models of sepsis-ALI. The pathological changes were evaluated by hematoxylin-eosin staining. Pulmonary vascular permeability was detected using wet-dry ratio, fluorescein isothiocyanate (FITC)-dextran, FITC-albumin, and immunoglobulin M concentration of bronchoalveolar lavage fluid. FITC-dextran and trans-endothelial electrical resistance assay were used to evaluate endothelial permeability on LPS-stimulated HUVECs. The mRNA expressions of TJ proteins were detected by real-time quantitative polymerase chain reaction. Then, protein levels were detected through Western blot analysis and immunofluorescence. The content of phosphatidic acid (PA), a downstream product of PLD2, was detected using an enzyme-linked immunosorbent assay kit. PLD2 deficiency not only alleviated lung histopathological changes and improved pulmonary vascular permeability but also increased the survival rate of ALI mice. Knockout of PLD2 or treatment with the PLD2 inhibitor can reduce the damage of endothelial TJ proteins, namely, claudin5, occludin and zonula occludens protein-1, in sepsis-ALI mice and LPS-stimulated HUVECs. The level of the PLD2 catalytic product PA increased in LPS-stimulated HUVECs, and exogenous PA can reduce the TJ protein expression and increase signal transducer and activator of transcription 3 (STAT3) phosphorylation in vitro. Inhibition of STAT3 phosphorylation attenuated PA-induced degradation of endothelial TJs. PLD2 knockout or inhibition may protect against LPS-induced lung injury by regulating the PA/STAT3 phosphorylation/endothelial TJ axis.

Advances in BBB on Chip and Application for Studying Reversible Opening of Blood-Brain Barrier by Sonoporation.

The complex structure of the blood-brain barrier (BBB), which blocks nearly all large biomolecules, hinders drug delivery to the brain and drug assessment, thus decelerating drug development. Conventional in vitro models of BBB cannot mimic some crucial features of BBB in vivo including a shear stress environment and the interaction between different types of cells. There is a great demand for a new in vitro platform of BBB that can be used for drug delivery studies. Compared with in vivo models, an in vitro platform has the merits of low cost, shorter test period, and simplicity of operation. Microfluidic technology and microfabrication are good tools in rebuilding the BBB in vitro. During the past decade, great efforts have been made to improve BBB penetration for drug delivery using biochemical or physical stimuli. In particular, compared with other drug delivery strategies, sonoporation is more attractive due to its minimized systemic exposure, high efficiency, controllability, and reversible manner. BBB on chips (BOC) holds great promise when combined with sonoporation. More details and mechanisms such as trans-endothelial electrical resistance (TEER) measurements and dynamic opening of tight junctions can be figured out when using sonoporation stimulating BOC, which will be of great benefit for drug development. Herein, we discuss the recent advances in BOC and sonoporation for BBB disruption with this in vitro platform.

Calcium-dependent cAMP mediates the mechanoresponsive behaviour of endothelial cells to high-frequency nanomechanostimulation

The endothelial junction plays a central role in regulating intravascular and interstitial tissue permeability. The ability to manipulate its integrity therefore not only facilitates an improved understanding of its underlying molecular mechanisms but also provides insight into potential therapeutic solutions. Herein, we explore the effects of short-duration nanometer-amplitude MHz-order mechanostimulation on interendothelial junction stability and hence the barrier capacity of endothelial monolayers. Following an initial transient in which the endothelial barrier is permeabilised due to Rho–ROCK-activated actin stress fibre formation and junction disruption typical of a cell's response to insults, we observe, quite uniquely, the integrity of the endothelial barrier to not only spontaneously recover but also to be enhanced considerably—without the need for additional stimuli or intervention. Central to this peculiar biphasic response, which has not been observed with other stimuli to date, is the role of second messenger calcium and cyclic adenosine monophosphate (cAMP) signalling. We show that intracellular Ca2+, modulated by the high frequency excitation, is responsible for activating reorganisation of the actin cytoskeleton in the barrier recovery phase, in which circumferential actin bundles are formed to stabilise the adherens junctions via a cAMP-mediated Epac1–Rap1 pathway. Despite the short-duration stimulation (8 min), the approximate 4-fold enhancement in the transendothelial electrical resistance (TEER) of endothelial cells from different tissue sources, and the corresponding reduction in paracellular permeability, was found to persist over hours. The effect can further be extended through multiple treatments without resulting in hyperpermeabilisation of the barrier, as found with prolonged use of chemical stimuli, through which only 1.1- to 1.2-fold improvement in TEER has been reported. Such an ability to regulate and enhance endothelial barrier capacity is particularly useful in the development of in vitro barrier models that more closely resemble their in vivo counterparts.

Impaired barrier integrity of endothelial cells induced by PEGylated black phosphorus nanosheets

PEGylated black phosphorus nanosheets (PEG-BPNSs) have shown promising applications in biomedicine and potentially interact with the vasculature following iatrogenic exposures. Whether the exposure to PEG-BPNSs could induce toxic effects on endothelial cells that line the blood vessels remains largely unknown. Herein, we investigate the cellular response and transcriptional profiling of human umbilical vein endothelial cells (HUVECs) after the exposure to BPNSs and PEG-BPNSs. BPNSs and PEG-BPNSs induce cellular elongation and cause significant cytotoxicity to HUVECs at 0.8 μg/mL, with viabilities of 87.8% and 87.7% respectively. The transcriptome analysis indicates that BPNSs and PEG-BPNSs at 0.4 μg/mL cause marked alterations in the expression of genes associated with detection of stimulus, ion transmembrane transport and components of plasma membrane. BPNSs and PEG-BPNSs at 0.4 μg/mL decrease the transendothelial electrical resistance (TEER) across monolayers of HUVECs by 22.8% and 20.3% compared to the control, respectively. The disturbance of tight junctions (TJs) after 24 h exposure to 0.4 μg/mL BPNSs and PEG-BPNSs is indicated with the downregulated mRNA expression of zona occluden-1 (ZO-1) by respective 16.5% and 29.9%, which may be involved in the impairment of endothelial barrier integrity. Overall, the response of HUVECs to PEG-BPNSs and BPNSs has no statistical difference, suggesting that PEGylation does not attenuate the BPNSs-induced endothelial injury. This study demonstrates the detrimental effects of BPNSs and PEG-BPNSs on barrier integrity of HUVECs, contributing to our understanding on the potential toxicological mechanisms.

The contribution of hyperhomocysteinemia and hypoperfusion to amyloid‐beta induced cerebral endothelial cell dysfunction

AbstractBackgroundCerebrovascular dysfunction has been implicated as a major contributor to Alzheimer’s Disease (AD) pathology, with particularly endothelial cell (EC) stress promoting the focal ischemia, cerebral blood flow impairments, and blood brain barrier (BBB) permeability that are pathologically characteristic in AD. Recent evidence has emerged suggesting a link between cardiovascular (CV) diseases and AD pathology, particularly showing that CV/cerebrovascular risk factors, including hyperhomocysteinemia (Hhcy) and hypoperfusion (oxygen and glucose deprivation (OGD)), contribute to AD pathology and risk. Despite this, the underlying molecular mechanisms for this interaction remain unclear. Previously our lab has demonstrated that amyloid beta (Aβ), particularly Aβ40‐Q22 (the vasculotropic Dutch mutant), promotes TRAIL death receptor (DR)‐mediated apoptosis, barrier permeability, and angiogenic impairment within human cerebral ECs. We tested the hypothesis that Hhcy and hypoperfusion exacerbate Aβ‐induced cerebral EC TRAIL DR‐mediated apoptosis, barrier dysfunction, and angiogenesis defects.MethodHuman cerebral microvascular ECs were challenged with AβQ22 and/or homocysteine (Hcy) in the presence/absence of hypoperfusion. Apoptotic mediator expression, caspase activation, and DNA fragmentation were measured to assess apoptosis. BBB protein expression and trans‐endothelial‐electrical‐resistance (TEER) were measured to assess EC barrier integrity. Angiogenesis inhibition and activation assays were utilized to measure EC angiogenic capability.ResultAβQ22 and Hcy challenge independently upregulated EC expression of TRAIL DR‐related apoptotic mediators and caspase activity, and, at certain time‐points, resulted in an additive upregulation of apoptotic mediators and caspase activity. AβQ22, Hcy, and hypoperfusion individually increased DNA fragmentation within cerebral ECs. Combination treatments of AβQ22 and Hcy created an additive effect on DNA fragmentation. Hypoperfusion and AβQ22 independently decreased BBB protein expression and TEER, while an additive decrease was observed with the combined challenges. AβQ22 and Hcy independently decreased angiogenesis progression, with some timepoints revealing an additive decrease with the combination treatment.ConclusionThe presence of CV risk factors seems to exacerbate Aβ induced‐EC apoptosis, barrier dysfunction, and angiogenic impairment, revealing several of the specific molecular mechanism through which amyloidosis and CV risk factors may additively act to produce EC dysfunction and death in AD pathology.

Critical role of Rho proteins in myosin light chain di-phosphorylation during early phase of endothelial barrier disruption

We previously reported the Rho-associated coiled-coil containing protein kinase (ROCK)-mediated di-phosphorylation of myosin light chain (MLC) and actin bundle formation at the cell periphery as early events of the endothelial barrier disruption. We herein examined the role of RhoA during early events of barrier disruption. Treatment of cultured porcine aortic endothelial cells with simvastatin prevented the decrease in trans-endothelial electrical resistance, MLC di-phosphorylation and peripheral actin bundle formation seen 3 min after thrombin stimulation. Co-treatment with geranylgeranyl pyrophosphate rescued the thrombin-induced events. Thrombin increased a GTP-bound form of RhoA and phosphorylation of myosin phosphatase target subunit 1 (MYPT1) at the ROCK site. The intracellular introduction of the inhibitory protein of RhoA inhibited the thrombin-induced di-phosphorylation of MLC. However, knockdown of either one of RhoA, RhoB or RhoC failed to inhibit thrombin-induced MLC di-phosphorylation. The findings suggest that Rho proteins play a critical role during early events of thrombin-induced barrier disruption.

Contribution of astrocytic MMP9 toward progression of VCID pathology

AbstractBackgroundVascular contributions to cognitive impairment and dementia (VCID) is one of the leading causes of dementia. High levels of plasma homocysteine or hyperhomocysteinemia has been characterized as a risk factor for VCID however, the mechanism underlying the connection between hyperhomocysteinemia and development of VCID pathology remains elusive. I hypothesize that hyperhomocysteinemia initiates a pro‐inflammatory cascade that increases the activity of MMP9 causing both perivascular astrocytes to dissociate from their vessels and initiating the degradation of endothelial cell tight junction proteins, leading to blood brain barrier dysfunction and the progression toward VCID pathology.MethodFor in vivo studies, C57BL6 WT and MMP9KO mice were placed on a control diet or a diet deficient in folate, vitamins B6 and B12 and enriched in methionine to induce hyperhomocysteinemia for 4, 8, 12, and 16 weeks. For in vitro, experiments, primary astrocytes from WT and MMP9KO mice were treated with a 50μM homocysteine stimulus or an inflammatory stimulus (TNF‐a, IL‐1b and C1q) for 48 hours and the conditioned media was collected. This homocysteine or inflammatory astrocyte conditioned media was then used to treat WT endothelial cells. Immunohistochemistry and gene expression analysis were used to determine neuroinflammatory changes while histology was used to identify changes in astrocytic end‐feet proteins, tight junction proteins, vascular density and microhaemorrhages. Both gel and in situ zymography were used to assess proteinase activity of MMP9 and related gelatinases. Western blots were used to investigate substrates of MMP9 including claudin, occludin and β‐dystroglycan. Behaviour was assessed using rotarod, novel object recognition and spontaneous alternation testing. Transendothelial electrical resistance and sodium fluorescein assays were used to measure the integrity of endothelial cell tight junctions in vitro.ResultStudies are underway to examine changes in cognition, microhaemorrhages, neuroinflammation, astrocyte end‐foot integrity, tight junction protein integrity and activity of MMP9.ConclusionCollectively, our findings suggest that astrocytic MMP9 may play an integral role in the mechanism associating homocysteine induced neuroinflammation with vascular pathogenesis leading to VCID, highlighting this pathway as an important subject for future study.

A human-derived neurovascular unit in vitro model to study the effects of cellular cross-talk and soluble factors on barrier integrity.

The blood-brain barrier (BBB) restricts paracellular and transcellular diffusion of compounds and is part of a dynamic multicellular structure known as the "neurovascular unit" (NVU), which strictly regulates the brain homeostasis and microenvironment. Several neuropathological conditions (e.g., Parkinson's disease and Alzheimer's disease), are associated with BBB impairment yet the exact underlying pathophysiological mechanisms remain unclear. In total, 90% of drugs that pass animal testing fail human clinical trials, in part due to inter-species discrepancies. Thus, in vitro human-based models of the NVU are essential to better understand BBB mechanisms; connecting its dysfunction to neuropathological conditions for more effective and improved therapeutic treatments. Herein, we developed a biomimetic tri-culture NVU in vitro model consisting of 3 human-derived cell lines: human cerebral micro-vascular endothelial cells (hCMEC/D3), human 1321N1 (astrocyte) cells, and human SH-SY5Y neuroblastoma cells. The cells were grown in Transwell hanging inserts in a variety of configurations and the optimal setup was found to be the comprehensive tri-culture model, where endothelial cells express typical markers of the BBB and contribute to enhancing neural cell viability and neurite outgrowth. The tri-culture configuration was found to exhibit the highest transendothelial electrical resistance (TEER), suggesting that the cross-talk between astrocytes and neurons provides an important contribution to barrier integrity. Lastly, the model was validated upon exposure to several soluble factors [e.g., Lipopolysaccharides (LPS), sodium butyrate (NaB), and retinoic acid (RA)] known to affect BBB permeability and integrity. This in vitro biological model can be considered as a highly biomimetic recapitulation of the human NVU aiming to unravel brain pathophysiology mechanisms as well as improve testing and delivery of therapeutics.

Functional Blood-Brain Barrier Model with Tight Connected Minitissue by Liquid Substrates Culture.

The functional blood-brain barrier (BBB) model can provide a reliable tool for better understanding BBB transport mechanisms and in vitro preclinical experimentation. However, recapitulating microenvironmental complexities and physiological functions in an accessible approach remains a major challenge. Here, a new BBB model with a high-cell spatial density and tightly connected biomimetic minitissue is presented. The minitissue, pivotal functional structure of the BBB model, is fabricated by a novel and easy-to-use liquid substrate culture (LSC) method, which allows cells to self-assemble and self-heal into macrosized, tightly connected membranous minitissue. The minitissue with uniform thickness can be easily harvested in their entirety with extracellular matrix. Attributed to the tightly connected minitissue formed by LSC, the fabricated BBB biomimetic model has 1 to 2 orders of magnitude higher transendothelial electric resistance than the commonly reported BBB model. It also better prevents the transmission of large molecular substances, recapitulating the functional features of BBB. Furthermore, the BBB biomimetic model provides feedback regarding BBB-destructive drugs, exhibits selective transmission, and shows efflux pump activity. Overall, this model can serve as an accessible tool for life science or clinical medical researchers to enhance the understanding of human BBB and expedite the development of new brain-permeable drugs.

Recombinant human plasma gelsolin reverses increased permeability of the blood–brain barrier induced by the spike protein of the SARS-CoV-2 virus

BackgroundPlasma gelsolin (pGSN) is an important part of the blood actin buffer that prevents negative consequences of possible F-actin deposition in the microcirculation and has various functions during host immune response. Recent reports reveal that severe COVID-19 correlates with reduced levels of pGSN. Therefore, using an in vitro system, we investigated whether pGSN could attenuate increased permeability of the blood–brain barrier (BBB) during its exposure to the portion of the SARS-CoV-2 spike protein containing the receptor binding domain (S1 subunit).Materials and methodsTwo- and three-dimensional models of the human BBB were constructed using the human cerebral microvascular endothelial cell line hCMEC/D3 and exposed to physiologically relevant shear stress to mimic perfusion in the central nervous system (CNS). Trans-endothelial electrical resistance (TEER) as well as immunostaining and Western blotting of tight junction (TJ) proteins assessed barrier integrity in the presence of the SARS-CoV-2 spike protein and pGSN. The IncuCyte Live Imaging system evaluated the motility of the endothelial cells. Magnetic bead-based ELISA was used to determine cytokine secretion. Additionally, quantitative real-time PCR (qRT-PCR) revealed gene expression of proteins from signaling pathways that are associated with the immune response.ResultspGSN reversed S1-induced BBB permeability in both 2D and 3D BBB models in the presence of shear stress. BBB models exposed to pGSN also exhibited attenuated pro-inflammatory signaling pathways (PI3K, AKT, MAPK, NF-κB), reduced cytokine secretion (IL-6, IL-8, TNF-α), and increased expression of proteins that form intercellular TJ (ZO-1, occludin, claudin-5).ConclusionDue to its anti-inflammatory and protective effects on the brain endothelium, pGSN has the potential to be an alternative therapeutic target for patients with severe SARS-CoV-2 infection, especially those suffering neurological complications of COVID-19.

Antioxidant Activity of Cyanidin-3-O-Glucoside and Verbascoside in an in Vitro Model of Diabetic Retinopathy.

Reactive oxygen species (ROS) accumulation plays a pivotal role in the onset of cell damage induced by hyperglycemia and represents one of the major factors in the pathogenesis of diabetic retinopathy. In this study, we tested the antioxidants cyanidin-3-O-glucoside (C3G) and verbascoside (Verb) in the protection of retinal endothelium against glucose toxicity "in vitro". Increasing amounts (5-50 μM) of C3G, Verb or the combination of both compounds were tested in Human Retinal Endothelial Cells (HREC) grown with normal glucose (5 mM, NG) or high glucose (25 mM, HG). Reduced cell viability and enhanced ROS levels (evaluated by MTT and H2DCFDA assays, respectively) in HG-stimulated HREC were restored by C3G and Verb in a dose-dependent manner, achieving the maximum protection in the presence of both compounds. Moreover, co-treatment with C3G and Verb worked better than each single molecule alone in the prevention of the disruption of blood-retinal-barrier-like properties by HG in a confluent HREC monolayer, as assessed by trans endothelial electrical resistance (TEER) and Na-Fluorescein permeability assays. Accordingly, C3G and Verb together also better counteracted the HG-induced down-regulation of the tight junction membrane proteins Zonula Occludens-1 and VE-Cadherin evaluated by immunocytochemical and Western blot analyses. In conclusion, our data indicate that C3G and Verb could efficiently protect the retinal endothelium against high glucose damage.

Hsa_circ_0074158 regulates the endothelial barrier function in sepsis and its potential value as a biomarker.

Background: Sepsis is one of the main causes of death in critically ill patients with high morbidity and mortality. Circular RNAs (CircRNAs) are aberrantly expressed, and play significant regulatory roles in many diseases. However, the expression profiles and functions of circRNAs in sepsis have not yet been fully clarified. Methods: Our present study performed an RNA sequencing (RNA-seq) analysis to assess the expression profiles of circRNAs in vitro. We applied the quantitative real-time polymerase chain reaction (RT-qPCR) to verify the RNA-seq results. The analyses of Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, the competitive endogenous RNA (ceRNA) regulatory networks, were performed to explore the potential mechanism in sepsis. And then, significantly up-regulated differentially expressed (DE) circRNA, hsa_circ_0074158, was selected for further study. Hsa_circ_0074158 was silenced to investigate its regulatory function in sepsis, and the barrier function was also examined in vitro. Endothelial cell junctions were valued using Vascular endothelial cadherin (VE-cadherin), which was detected by immunofluorescence staining. We measured endothelial permeability by transendothelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)-dextran extravasation. Results: In total, 203 significantly DE circRNAs, including 77 up-regulated and 126 down-regulated, were identified. In vitro, the RT-qPCR assay showed that the expression pattern of hsa_circ_0074158, hsa_circ_RSBN1L_11059, hsa_circ_0004188, and hsa_circ_0005564 were consistent with the results from RNA-seq analysis. The expression of hsa_circ_0074158 detected by RT-qPCR in vivo was also consistent with the RNA-seq results. The ceRNA networks, GO enrichment, and the KEGG pathway analyses revealed that circRNAs may be related to the barrier function in sepsis. The immunofluorescence assay showed that the suppression of hsa_circ_0074158 expression significantly enhanced the expression of VE-cadherin, which was suppressed in lipopolysaccharide (LPS)-induced sepsis. Additionally, hsa_circ_0074158 knockdown could partially reverse the LPS-induced TEER reduction and FITC-dextran extravasation elevation in sepsis. Conclusion: In conclusion, we have found DE circRNAs could serve as potential biomarkers and therapeutic targets for sepsis. Hsa_circ_0074158 plays a vital role in sepsis and is related to the disruption of the endothelial barrier.

Abstract 15915: Ultrasound Targeted Microbubble Cavitation-Induced Endothelial Hyperpermeability is Calcium-Dependent and Regulated by Enos Signaling

Introduction: Ultrasound ( US )-targeted microbubble ( MB ) cavitation ( UTMC ) facilitates delivery of cell-impermeant drugs across the endothelial barrier; mechanisms are poorly understood. We reported that UTMC causes inter-endothelial gaps in cultured endothelial cell ( EC ) monolayers, which could increase barrier permeability, and that UTMC increases Ca 2+ influx into ECs. Given that increased intracellular Ca 2+ activates eNOS, and nitric oxide (NO) release promotes microvascular permeability, we hypothesized that UTMC increases intracellular Ca 2+ through activation of mechanosensitive Piezo-1 Ca 2+ channels, leading to NO synthesis and endothelial barrier hyperpermeability. Methods: Transwells were endothelialized with human coronary artery ECs. Lipid MBs were added, and US was delivered for 10 s (1 MHz, 250 kPa, 10 cycles, 10 ms interval). Permeability was measured using dextran transfer and transendothelial electrical resistance ( TEER ). GsMTx4 and L-NAME were used to inhibit Piezo1 and eNOS, respectively; Fluo-4 AM to visualize Ca 2+ influx; DAF-FM to detect NO. S-nitrosylation was measured with biotin switch assay. Results: UTMC reduced TEER (1.7 fold, p&lt;0.05 ) and increased dextran flux across the transwell (2-fold, p&lt;0.01 for 70 kDa dextran, 1.6 fold, p&lt;0.05 for 10 kDa dextran), which was abrogated by L-NAME. UTMC significantly increased Ca 2+ influx into ECs and tripled NO ( p&lt;0.05 ), but these increases and UTMC-mediated hyperpermeability were blunted by Piezo1 channel inhibition ( p&lt;0.0001) . UTMC increased S-nitrosylation of proteins (1.7 fold, p&lt;0.01 ) and was associated with s-nitrosylation of VE-cadherin, its redistribution into focal adhesion junctions, and increased stress fiber formation, which were abolished by L-NAME. Conclusions: UTMC causes endothelial barrier hyperpermeability through a Ca 2+ -dependent mechanism, where Ca 2+ influx into ECs, mediated in part by mechanosensitive Piezo1 channels, modulates eNOS signaling. Our data suggest that eNOS regulates UTMC-induced hyperpermeability, possibly through s-nitrosylation of VE-cadherin. Further studies to examine mechanisms regulating UTMC bioeffects will aid in clinical translation and optimization of UTMC for delivery of cell-impermeant drugs.