Monolayer Permeability Research Articles

Effect of Platelet-Activating Factor on Barrier Function of ARPE-19 Cells.

AimTo examine the effects of platelet-activating factor (PAF) on the barrier functions of cultured retinal pigment epithelial (RPE) cells.MethodsA human RPE cell line (ARPE-19) was cultured on microporous filter supports and treated with PAF and WEB 2086, a specific PAF-receptor (PAF-R) antagonist. The permeability of the RPE monolayer was measured using transepithelial electrical resistance (TER) and sodium fluorescein flux. The expression of the tight junction protein zonula occludens (ZO)-1 and the adherens junction protein N-cadherin was assessed using immunohistochemistry and Western blotting. We also measured the vascular endothelial growth factor (VEGF) concentrations in PAF-treated cultures and re-measured RPE monolayer permeability in the presence of VEGF-neutralizing antibodies.ResultsPAF significantly decreased the TER and enhanced the sodium fluorescein flux of the RPE monolayer and downregulated the expression of ZO-1 and N-cadherin. These effects were abolished by WEB 2086-mediated blockage of the PAF-R. PAF stimulation increased VEGF expression in RPE cells, and the antibody-mediated neutralization of VEGF caused a partial recovery of the barrier properties.ConclusionThe barrier functions of ARPE-19 cells were altered by PAF, and these effects were partly mediated by an upregulation of VEGF expression in these cells. Our results contribute to the growing body of evidence supporting the role of PAF in choroidal neovascularization. Our findings suggest that PAF is a novel target in the development of therapies for increased permeability of the RPE monolayer.

Regulation of the thrombin/protease-activated receptor 1 axis by chemokine (CXC motif) receptor 4

The chemokine receptor CXCR4, a G protein-coupled receptor (GPCR) capable of heteromerizing with other GPCRs, is involved in many processes, including immune responses, hematopoiesis, and organogenesis. Evidence suggests that CXCR4 activation reduces thrombin/protease-activated receptor 1 (PAR1)-induced impairment of endothelial barrier function. However, the mechanisms underlying cross-talk between CXCR4 and PAR1 are not well-understood. Using intermolecular bioluminescence resonance energy transfer and proximity ligation assays, we found that CXCR4 heteromerizes with PAR1 in the HEK293T expression system and in human primary pulmonary endothelial cells (hPPECs). A peptide analog of transmembrane domain 2 (TM2) of CXCR4 interfered with PAR1:CXCR4 heteromerization. In HTLA cells, the presence of CXCR4 reduced the efficacy of thrombin to induce β-arrestin-2 recruitment to recombinant PAR1 and enhanced thrombin-induced Ca2+ mobilization. Whereas thrombin-induced extracellular signal-regulated protein kinase 1/2 (ERK1/2) phosphorylation occurred more transiently in the presence of CXCR4, peak ERK1/2 phosphorylation was increased when compared with HTLA cells expressing PAR1 alone. CXCR4-associated effects on thrombin-induced β-arrestin-2 recruitment to and signaling of PAR1 could be reversed by TM2. In hPPECs, TM2 inhibited thrombin-induced ERK1/2 phosphorylation and activation of Ras homolog gene family member A. CXCR4 siRNA knockdown inhibited thrombin-induced ERK1/2 phosphorylation. Whereas thrombin stimulation reduced surface expression of PAR1, CXCR4, and PAR1:CXCR4 heteromers, chemokine (CXC motif) ligand 12 stimulation reduced surface expression of CXCR4 and PAR1:CXCR4 heteromers, but not of PAR1. Finally, TM2 dose-dependently inhibited thrombin-induced impairment of hPPEC monolayer permeability. Our findings suggest that CXCR4:PAR1 heteromerization enhances thrombin-induced G protein signaling of PAR1 and PAR1-mediated endothelial barrier disruption.

The Synergistic and Neuroprotective Effects of Alcohol-Antioxidant Treatment on Blood-Brain Barrier Endothelial Cells.

Alcohol (EtOH) is reported to adversely affect one of the most crucial roles of the blood-brain barrier (BBB), the regulation of its permeability, thereby compromising the stability of the homeostatic environment of the brain. The central component of the BBB, endothelial cells (ECs), regulates BBB transcellular transport, while their paracellular pathways are made virtually impermeable by molecular structures called tight junctions (TJs). These TJs are composed of proteins, such as claudin-5, a protein involved in the regulation of paracellular permeability and of key interest in this study. The working hypothesis of this study postulated that the high levels of antioxidants (AOs) in the fermented Aspalathus linearis (Rooibos; Rf) tincture may protect the ECs of the BBB against oxidative stress induced by EtOH exposure. Cells were exposed for 24hours to selected concentrations of EtOH (25 and 100mM), Rf (containing an antioxidant equivalence of 1.9nM Aspalathin), and cotreatments of EtOH and Rf. Cell viability, live cell number, and toxicity were analyzed using the trypan blue exclusion assay. RT-qPCR was implemented to quantify claudin-5 transcription. In addition, permeability (Transepithelial Electrical Resistance) of bEnd5 monolayers was measured. The experimental timeline for the above-mentioned parameters was 24 and 48hours. Our study showed that simultaneous exposure of Rf and EtOH was able to negate the effects of EtOH on cell viability and cell proliferation, but was not able to reverse or reduce the effects of EtOH on claudin-5 transcription and paracellular permeability. Furthermore, a novel finding in this study suggests that very low concentrations of AOs in tinctures such as Rooibos tea could profoundly alter the redox status of brain ECs.

Gut-lymph-lung pathway mediates sepsis-induced acute lung injury.

This review attempts to unveil the possible mechanisms underlying how gut lymph affects lung and further gives rise to acute respiratory distress syndrome, as well as potential interventional targets under the condition of ischemia-reperfusion injury. We searched electronic databases including PubMed, MEDLINE, Cochrane Central Register of Controlled Trials, Google Scholar, Web of Science, and Embase to identify relevant literatures published up to December 2019. We enrolled the literatures including the Mesh Terms of “gut lymph or intestinal lymph and acute lung injury or acute respiratory distress syndrome.” Gut is considered to be the origin of systemic inflammation and the engine of multiple organ distress syndrome in the field of critical care medicine, whereas gut lymph plays a pivotal role in initiation of ischemia-reperfusion injury-induced acute respiratory distress syndrome. In fact, in the having been established pathologic model of sepsis leading to multiple organ dysfunction named by Gut Lymph theory, a variety of literatures showed the position and role of changes in gut lymph components in the initiation of systemic inflammatory response, which allows us to screen out potential intervention targets to pave the way for future clinic and basic research.

Efficacy of theobromine in preventing intestinal CaCo-2 cell damage induced by oxysterols

The alteration of the intestinal barrier function is currently believed to be involved in the pathogenesis of gut diseases mainly associated with the activation of inflammation processes.Diet plays an important role in the control of human gut integrity. Theobromine is a natural methylxanthine present in dark chocolate particularly abundant in cocoa bean shell. This is a polyphenol rich by-product generated in cocoa industrial processing, which is gaining value as a functional ingredient. This study aims to highlight for the first time the capability of theobromine in protecting the intestinal cell monolayer from a mixture of dietary oxysterols showing an inflammatory action in terms of IL-8 and MCP-1 overproduction. Differentiated CaCo-2 cells were treated with 60 μM oxysterol mixture and pre-incubated with 10 μM theobromine. Intestinal barrier damage was investigated in terms of tight junction claudin 1, occludin and JAM-A protein levels, matrix metalloproteinase (MMP) −2 and −9 activation and anti/pro-apoptotic protein changes.The observed cell monolayer permeability protection by theobromine may be due to its ability to inhibit the production of cytokines and MMPs that can be responsible for tight junction loss and apoptosis in intestinal cells. Our findings provide additional mechanistic hints on the healthy effect of theobromine cocoa component as an attractive natural molecule in the prevention of inflammatory gut diseases.

Serum amyloid A-induced blood-brain barrier dysfunction associated with decreased claudin-5 expression in rat brain endothelial cells and its inhibition by high-density lipoprotein in vitro

The blood-brain barrier (BBB) is the multicellular interface located between the peripheral circulation and the brain parenchyma. BBB dysfunction is reported in many CNS diseases, such cognitive impairment, depression, Alzheimer’s disease (AD), and multiple sclerosis (MS). Emerging evidence indicates that liver-derived inflammatory mediators are upregulated in neurological diseases with BBB dysfunction. Serum amyloid A (SAA), an acute phase protein secreted by hepatocytes, could be a candidate inflammatory signaling molecule transmitted from the liver to the brain; however, its contribution to BBB dysfunction is poorly understood. The present study aimed to elucidate the involvement of SAA in BBB impairment in an in vitro BBB model using rat brain microvascular endothelial cells (RBECs). We demonstrated that Apo-SAA significantly decreased transendothelial electrical resistance (TEER) and increased sodium fluorescein (Na-F) permeability in RBEC monolayers. Apo-SAA also decreased claudin-5 expression levels in RBECs. Furthermore, the Apo-SAA-mediated impairment of the BBB with decreased claudin-5 expression was inhibited by the addition of a high-density lipoprotein (HDL) related to SAA in plasma. These findings suggest that HDL counteracts the effects of SAA on BBB function. Therefore, the functional imbalance between SAA and HDL may induce BBB impairment, thereby triggering development of neuroinflammation. SAA could be a significant endogenous mediator in the liver-to-brain inflammation axis.

Structural, Functional, and Metabolic Alterations in Human Cerebrovascular Endothelial Cells during Toxoplasma gondii Infection and Amelioration by Verapamil In Vitro.

Toxoplasma gondii (T. gondii), the causative agent of toxoplasmosis, is a frequent cause of brain infection. Despite its known ability to invade the brain, there is still a dire need to better understand the mechanisms by which this parasite interacts with and crosses the blood–brain barrier (BBB). The present study revealed structural and functional changes associated with infection and replication of T. gondii within human brain microvascular endothelial cells (BMECs) in vitro. T. gondii proliferated within the BMECs and disrupted the integrity of the cerebrovascular barrier through diminishing the cellular viability, disruption of the intercellular junctions and increasing permeability of the BMEC monolayer, as well as altering lipid homeostasis. Proton nuclear magnetic resonance (1H NMR)-based metabolomics combined with multivariate data analysis revealed profiles that can be attributed to infection and variations in the amounts of certain metabolites (e.g., amino acids, fatty acids) in the extracts of infected compared to control cells. Notably, treatment with the Ca2+ channel blocker verapamil rescued BMEC barrier integrity and restricted intracellular replication of the tachyzoites regardless of the time of treatment application (i.e., prior to infection, early- and late-infection). This study provides new insights into the structural and functional changes that accompany T. gondii infection of the BMECs, and sheds light upon the ability of verapamil to inhibit the parasite proliferation and to ameliorate the adverse effects caused by T. gondii infection.

Lychee seed polyphenol protects the blood-brain barrier through inhibiting Aβ(25-35)-induced NLRP3 inflammasome activation via the AMPK/mTOR/ULK1-mediated autophagy in bEnd.3 cells and APP/PS1 mice.

Blood-brain barrier (BBB) dysfunction has been implicated in Alzheimer's disease (AD) and is closely linked to the release of proinflammatory cytokines in brain capillary endothelial cells. We have previously reported that lychee seed polyphenols (LSP) exerted anti-neuroinflammatory effect. In this study, we aimed to explore the protective effect of LSP on BBB integrity. The monolayer permeability of bEnd.3 cells, and the mRNA level and protein expression of tight junction proteins (TJs), including Claudin 5, Occludin, and ZO-1, were examined. In addition, the inhibition of Aβ(25-35)-induced NLRP3 inflammasome activation, and the autophagy induced by LSP were investigated by detecting the expression of NLRP3, caspase-1, ASC, LC3, AMPK, mTOR, and ULK1. Furthermore, the cognitive function and the expression of TJs, NLRP3, caspase-1, IL-1β, and p62 were determined in APP/PS1 mice. The results showed that LSP significantly decreased the monolayer permeability and inhibited the NLRP3 inflammasome in Aβ(25-35)-induced bEnd3 cells. In addition, LSP induced autophagy via the AMPK/mTOR/ULK1 pathway in bEnd.3 cells, and improved the spatial learning and memory function, increased the TJs expression, and inhibited the expression of NLRP3, caspase-1, IL-1β, and p62 in APP/PS1 mice. Therefore, LSP protects BBB integrity in AD through inhibiting Aβ(25-35)-induced NLRP3 inflammasome activation via the AMPK/mTOR/ULK1-mediated autophagy.

Altered paracellular permeability in intestinal cell monolayer challenged with lipopolysaccharide: Modulatory effects of pterostilbene metabolites

Epithelial barrier alteration is a central event in the pathogenesis of inflammatory bowel diseases. Lipopolysaccharide, correlated to the pathogenesis of such pathologies, has been demonstrated to cause altered membrane permeability, through the disruption and/or relocation of tight junction proteins, following redox-sensitive mitogen-activated protein kinases (MAPKs) modulation. Pterostilbene and its metabolite pinostilbene are natural stilbenoids which may reach relevant concentrations at intestinal level, together with their glucuronide and sulfate metabolites. The aim of our study was to evaluate the ability of these compounds to inhibit lipopolysaccharide-induced toxic effects on intestinal cell monolayer integrity and to explore the mechanism of action. Caco-2 cells, differentiated as enterocytes, were treated with lipopolysaccharide following pretreatment with the phenolic compounds at 1 μM physiological concentration. Caco-2 monolayer's permeability was monitored with time, measuring the transepithelial electrical resistance. Tight junction proteins were assessed by western blotting and immunofluorescence in lipopolysaccharide-treated cells, in relation to MAPK p38 and ERK1/2 activation. Pretreatment with all the phenolic compounds significantly slowed lipopolysaccharide-induced transepithelial electrical resistance decrease, preserved tight junction proteins levels and reduced MAPKs phosphorylation. The reported findings indicate that pterostilbene and its metabolites may counteract lipopolysaccharide-induced alteration of epithelial permeability, one of the initial events in the intestinal inflammatory process.

Noncanonical EphA2 Signaling Is a Driver of Tumor-Endothelial Cell Interactions and Metastatic Dissemination in BRAF Inhibitor‒Resistant Melanoma

Acquired BRAF/MAPK/extracellular signal‒regulated kinase inhibitor resistance in melanoma results in a new transcriptional state associated with an increased risk of metastasis. In this study, we identified noncanonical ephrin receptor (Eph) EphA2 signaling as a driver of the resistance-associated metastatic state. We used mass spectrometry‒based proteomic and phenotypic assays to demonstrate that the expression of active noncanonical EphA2-S897E in melanoma cells led to a mesenchymal-to-amoeboid transition driven by Cdc42 activation. The induction of mesenchymal-to-amoeboid transition promoted melanoma cell invasion, survival under shear stress, adhesion to endothelial cells under continuous-flow conditions, increased permeability of endothelial cell monolayers, and stimulated melanoma transendothelial cell migration. Invivo, melanoma cells expressing EphA2-S897E or active Cdc42 showed superior lung retention after tail-vain injection. Analysis of BRAF inhibitor‒sensitive and ‒resistant melanoma cells demonstrated resistance to be associated with a mesenchymal-to-amoeboid transition switch, upregulation of Cdc42 activity, increased invasion, and transendothelial migration. The drug-resistant metastatic state was dependent on histone deacetylase 8 activity. Silencing of histone deacetylase 8 led to the inhibition of EphA2 and protein kinase B phosphorylation, reduced invasion, and impaired melanoma cell-endothelial cell interactions. In summary, we have demonstrated that the metastatic state associated with acquired BRAF inhibitor resistance is dependent on noncanonical EphA2 signaling, leading to increased melanoma-endothelial cell interactions and enhanced tumor dissemination.

HIF-1α Enhances Vascular Endothelial Cell Permeability Through Degradation and Translocation of Vascular Endothelial Cadherin and Claudin-5 in Rats With Burn Injury.

The mechanism underlying burn injury-induced enhanced vascular endothelial permeability and consequent body fluid extravasation is unclear. Here, the rat aortic endothelial cells (RAECs) were treated with the serum derived from rats with burn injury to elucidate the mechanism. Sprague-Dawley (SD) rats were grouped as follows (10 rats/group): control, 2, 4, 8, 12, and 24 hours postburn groups. The heart, liver, kidney, lung, jejunum, and ileum of rats injected with 2% Evans blue (EB) through the tail vein were excised to detect the EB level in each organ. The serum levels of hypoxia-inducible factor-1α (HIF-1α) and endothelin-1 (ET-1) were examined using enzyme-linked immunosorbent assay (ELISA). The effect of serum from 12-hour postburn group on the membrane permeability of RAEC monolayer, as well as on the mRNA and protein levels of ET-1, endothelin receptor A (ETA), ETB, and zonula occludens (ZO-1), was analyzed using quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. The membrane permeability of GV230/HIF-1α-transfected or shRNA-HIF-1α-transfected RAECs, as well as the expression levels of HIF-1α, ET-1, ETA, ETB, vascular endothelial (VE)-cadherin, and claudin-5, was analyzed using qRT-PCR and western blotting, whereas the localization of VE-cadherin and claudin-5 was examined using immunofluorescence. The serum HIF-1α and ET-1 levels in the burn groups, which peaked at 12 hours postburn, were significantly upregulated (P < .01) when compared with those in the control group. Additionally, the serum HIF-1α levels were positively correlated with vascular permeability. Compared with the shRNA-negative control-transfected RAECs, the shRNA-II/HIF-1α-transfected RAECs exhibited downregulated expression of HIF-1α, ET-1, ETA, and ETB (P < .01), and upregulated expression of ZO-1, claudin-5, and VE-cadherin (P < .05). Compared with the GV230-transfected RAECs, the GV230/HIF-1α-transfected RAECs exhibited upregulated expression of HIF-1α, ET-1, ETA, and ETB (P < .01), and downregulated expression of ZO-1, claudin-5, and VE-cadherin (P < .05). The GV230/HIF-1α-transfected RAECs exhibited degradation and translocation of VE-cadherin and claudin-5. In addition to degradation of VE-cadherin and claudin-5, HIF-1α mediated enhanced endothelial cell permeability through upregulation of ET-1, ETA, and ETB, and downregulation of ZO-1 and VE-cadherin in rats with burn injury.

LncRNA PVT1 exacerbates the inflammation and cell-barrier injury during asthma by regulating miR-149.

Asthma is a prevailing respiratory disease among children, characterized by allergic airway inflammation, airway remodeling, and airway hyperresponsiveness. Although it is well-known that long non-coding RNAs (lncRNAs) are linked to a variety of human diseases and well-documented, very few studies explore its role in asthma. In this study, we investigate the effects of lncRNA PVT1 on the promotion of airway inflammation and its associated mechanisms. Human small airway epithelial cells (HSAECs) with PVT1 overexpressed or knocked down were constructed, and platelet activating factor (PAF) was used to treat HSAECs to mimic the pathological process of asthma in vitro. The expressions of prostaglandin E2 (PGE2), interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α (TNF-α) were measured by enzyme-linked immunosorbent assay (ELISA). The expressions of PKC, MyD88, and NF-ĸB were measured by Western blot. Monolayer permeability of HSAECs was also compared within different groups. Luciferase reporter gene assay was employed to detect the targeting relationship between PVT1 and miR-149. The knockdown of PVT1 attenuated the levels of inflammatory factors induced by PAF and destruction of cell-barrier function. The overexpression of PVT1 facilitated the pathological development. Additionally, miR-149 was identified as a target microRNA of PVT1, and the overexpression of miR-149 could reverse the effects of PVT1 on PAF-induced HSAECs. These findings suggest that PVT1 may represent a novel potential target for treatment of asthma.

Caco-2 Cell Permeability of Flavonoids and Saponins from Gynostemma pentaphyllum: the Immortal Herb.

Gynostemma pentaphyllum (the immortal herb) has been an important component of Chinese Traditional Medicine for millennia. Recent clinical studies have revealed that the plant exhibits numerous beneficial biological activities, making it of interest to the pharmaceutical industry. An extract of the herb contains over 200 individual secondary metabolites including flavonol glycosides and dammarane saponins. To focus attention on the compounds most likely to be responsible for the biological activities, this study predicts the potential oral bioavailability of nine dammarane saponins and five flavonol glycosides from G. pentaphyllum using the Caco-2 cell monolayer permeability model. Two flavonoids, 8 and 9, and four saponins, 10, 11, 12, and 14, exhibited high permeability across the monolayers. The results indicated that a higher degree of glycosylation-facilitated permeability, suggestive of active transport. This study demonstrates the utility of the Caco-2 permeability assay as a method of identifying possible bioavailable compounds from medicinal herbal extracts.

Circulating Levels of Epirubicin Cause Endothelial Senescence While Compromising Metabolic Activity and Vascular Function

Anthracycline-based chemotherapy is a common treatment for cancer patients. Because it is delivered intravenously, endothelial cells are exposed first and to the highest concentrations, prior to diffusion to target cells. Not surprisingly, vascular dysfunction is a consequence of anthracycline therapy. While chemotherapy-induced endothelial damage at administration sites has been investigated, the effects of lower doses encountered by distant microvascular networks has not. The aim of this study was to investigate the impact of epirubicin, a widely used anthracycline, on healthy endothelial cells to elucidate its effects on microvascular physiology. Here, endothelial cells were briefly exposed to low doses of epirubicin to recapitulate levels in circulation following dilution in the blood and compound half-life in circulation. Both immediate and prolonged responses to treatment were assessed to determine changes in endothelial function. Epirubicin caused a decrease in proliferation and viability in hUVEC, with lower doses resulting in a senescent phenotype in a large proportion of cells, accompanied by a significant increase in pro-inflammatory cytokines and a significant decrease in metabolic activity. Epirubicin exposure also impaired endothelial function with delayed wound closure, reduced angiogenic potential and increased monolayer permeability downstream of VE-cadherin internalization. Primary lung endothelial cells obtained from epirubicin-treated mice similarly demonstrated reduced viability and functional impairment. In vivo, epirubicin treatment resulted in persistent reduction in lung vascular density and significantly increased infiltration of myeloid cells. Modulation of endothelial status and inflammatory tissue microenvironment observed in response to low doses of epirubicin may predict risk for long-term secondary pathologies associated with chemotherapy.

Suppression of Connexin 43 Leads to Strial Vascular Hyper-Permeability, Decrease in Endocochlear Potential, and Mild Hearing Loss.

Objective: Connexin 43 (Cx43) is a protein constituent of gap junctions (GJs) in various barrier cells, especially astrocytes and microglia of the blood-brain-barrier (BBB), where it plays an important role in intercellular communication and regulation of the barrier. Despite the importance of Cx43 in other blood barriers, not much attention has been paid to expression and function of Cx43 in the blood-labyrinth-barrier (BLB) of the stria vascularis in the cochlea.Methods: We used multiple research approaches, including immunocytochemical staining, patch-clamp dye loading technique, real-time quantitative reverse transcription (RT)-PCR, western blot, measurement of endocochlear potential (EP) with an electrode through the scala media, and auditory brainstem response to test hearing function.Results: We found Cx43 expressed in vascular endothelial cells (ECs) and perivascular resident macrophages (PVMs) in the stria vascularis of adult C57BL/6 mouse cochleae. In particular, we found Cx43 expressed in foot processes of PVMs at points of contact with the endothelium. Consistent with Cx43 expression in vivo, we also found Cx43 expressed in EC-EC and EC-PVM interfaces in a co-cultured cell line model. Using a patch-clamp dye loading technique, we demonstrated that Alexa Fluor® 568 dye injected into PVMs diffuses to connected neighboring ECs. The functional coupling between the ECs and PVMs is blocked by 18α-Glycyrrhetinic acid (18α-GA), a GJ blocker. Suppression of Cx43 with small interfering RNA (siRNA) in vivo significantly elevated hearing threshold and caused the EP to drop and the blood barrier to become more permeable. In further study, using in vitro primary EC cell line models, we demonstrated that suppression of Cx43 disrupts intercellular tight junctions (TJs) in the EC monolayer and increases endothelial monolayer permeability.Conculsion: Taken together, these findings underscore the importance of Cx43 expression in the normal ear for maintaining BLB integrity, normal EP, and hearing function.

Reparative effect of mesenchymal stromal cells on endothelial cells after hypoxic and inflammatory injury

BackgroundThe renal endothelium is a prime target for ischemia-reperfusion injury (IRI) during donation and transplantation procedures. Mesenchymal stromal cells (MSC) have been shown to ameliorate kidney function after IRI. However, whether this involves repair of the endothelium is not clear. Therefore, our objective is to study potential regenerative effects of MSC on injured endothelial cells and to identify the molecular mechanisms involved.MethodsHuman umbilical vein endothelial cells (HUVEC) were submitted to hypoxia and reoxygenation and TNF-α treatment. To determine whether physical interaction or soluble factors released by MSC were responsible for the potential regenerative effects of MSC on endothelial cells, dose-response experiments were performed in co-culture and transwell conditions and with secretome-deficient MSC.ResultsMSC showed increased migration and adhesion to injured HUVEC, mediated by CD29 and CD44 on the MSC membrane. MSC decreased membrane injury marker expression, oxidative stress levels, and monolayer permeability of injured HUVEC, which was observed only when allowing both physical and paracrine interaction between MSC and HUVEC. Furthermore, viable MSC in direct contact with injured HUVEC improved wound healing capacity by 45% and completely restored their angiogenic capacity. In addition, MSC exhibited an increased ability to migrate through an injured HUVEC monolayer compared to non-injured HUVEC in vitro.ConclusionsThese results show that MSC have regenerative effects on injured HUVEC via a mechanism which requires both physical and paracrine interaction. The identification of specific effector molecules involved in MSC-HUVEC interaction will allow targeted modification of MSC to apply and enhance the therapeutic effects of MSC in IRI.

Polysaccharide from the seeds of Plantago asiatica L. alleviates nonylphenol induced intestinal barrier injury by regulating tight junctions in human Caco-2 cell line

The intestinal epithelium is known as an important barrier to protect the body from harmful pathogens or toxic substance that may induce intestinal barrier injury. The aim of this study was to investigate the effects of polysaccharide from the seeds of Plantago asiatica L. (PLP) on nonylphenol (NP) induced intestinal barrier injury in vitro. Caco-2 cells were pretreated with PLP, or co-cultured with PLP and NP simultaneously, and cytotoxicity, LDH leakage, transepithelial electrical resistance (TEER), FITC-dextran flux and tight junction (TJ) proteins were conducted to evaluate the intestinal barrier function. The results suggested that PLP pretreatment or co-culture with NP could significantly attenuated NP induced Caco-2 cytotoxicity, suppressed LDH release, restored the TEER value and paracellular permeability of Caco-2 monolayers, which were attributed to enhancing the TJ protein expressions. In addition, PLP co-cultured with NP possessed better protective effects against NP induced cytotoxicity. This study indicated that PLP assuaged NP induced intestinal barrier injury by increasing TJ, and threw light on the development of a dietary supplementation for preventing exogenous toxic substances induced intestinal barrier injury or improving intestinal TJ barrier function.

Barrier Protection and Recovery Effects of Gut Commensal Bacteria on Differentiated Intestinal Epithelial Cells In Vitro.

Alterations in the gut microbiota composition play a crucial role in the pathogenesis of inflammatory bowel disease (IBD) as specific commensal bacterial species are underrepresented in the microbiota of IBD patients. In this study, we examined the therapeutic potential of three commensal bacterial species, Faecalibacterium prausnitzii (F. prausnitzii), Roseburia intestinalis (R. intestinalis) and Bacteroides faecis (B. faecis) in an in vitro model of intestinal inflammation, by using differentiated Caco-2 and HT29-MTX cells, stimulated with a pro-inflammatory cocktail consisting of interleukin-1β (IL-1β), tumor necrosis factor-α (TNFα), interferon-γ (IFNγ), and lipopolysaccharide (LPS). Results obtained in this work demonstrated that all three bacterial species are able to recover the impairment of the epithelial barrier function induced by the inflammatory stimulus, as determined by an amelioration of the transepithelial electrical resistance (TEER) and the paracellular permeability of the cell monolayer. Moreover, inflammatory stimulus increased claudin-2 expression and decreased occludin expression were improved in the cells treated with commensal bacteria. Furthermore, the commensals were able to counteract the increased release of interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) induced by the inflammatory stimulus. These findings indicated that F. prausnitzii, R. intestinalis and B. faecis improve the epithelial barrier integrity and limit inflammatory responses.

Preeclampsia serum increases CAV1 expression and cell permeability of human renal glomerular endothelial cells via down-regulating miR-199a-5p, miR-199b-5p, miR-204

IntroductionTo gain insight into mechanisms of preeclampsia (PE)-dependent proteinuria, this study focused on whether preeclampsia serum (PES) could induce hyperpermeability in human renal glomerular endothelial cells (HRGECs) via the miRNAs-Caveolin-1 (CAV-1)-dependent pathway. MethodsBioinformatics approach was used to identify miRNAs targeting CAV1. Normal pregnancy serum (NPS) and severe PES were used to treat HRGECs monolayer to demonstrate if PES could induce the expression of identified miRNAs. A luciferase reporter assay was used to determine whether CAV1 was a direct target of miR-199a-5p, miR-199b-5p, and miR-204. The relationship between the expression of miR-199a-5p, miR-199b-5p, miR-204, and CAV1 in HRGECs was determined using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. The gain-of-function and loss‐of‐function experiments were performed on HRGECs to investigate the effects of miR-199a-5p, miR-199b-5p, miR-204 on HRGECs permeability. ResultsWe identified that CAV1 3′UTR has putative binding sites for miR-199a-5p, miR-199b-5p, and miR-204, whereas miR-199a-5p does not appear to be a direct regulator of CAV1. We detected that PE serum downregulated the expression of miR-199a-5p, miR-199b-5p and miR-204, increased expression of CAV1 and increased cell monolayer permeability in HRGECs. The level of CAV1 and permeability decreased when miR-199b-5p or miR-204, but not miR-199a-5p, were overexpressed. DiscussionmiR-199b-5p and miR-204 may play a role in PES-induced increasing permeability of HRGECs by regulating CAV1 expression.

Tight junction protein claudin-1 is downregulated by TGF-β1 via MEK signaling in benign prostatic epithelial cells.

Benign prostatic hyperplasia (BPH) is arguably the most common disease in aging men. Although the etiology is not well understood, chronic prostatic inflammation is thought to play an important role in BPH initiation and progression. Our recent studies suggest that the prostatic epithelial barrier is compromised in glandular BPH tissues. The proinflammatory cytokine transforming growth factor beta 1 (TGF-β1) impacts tight junction formation, enhances epithelial barrier permeability, and suppresses claudin-1 messenger RNA expression in prostatic epithelial cells. However, the role of claudin-1 in the prostatic epithelial barrier and its regulation by TGF-β1 in prostatic epithelial cells are not clear. The expression of claudin-1 was analyzed in 22 clinical BPH specimens by immunohistochemistry. Human benign prostate epithelial cell lines BPH-1 and BHPrE1 were treated with TGF-β1 and transfected with small interfering RNAs specific to claudin-1. Epithelial monolayer permeability changes in the treated cells were measured using trans-epithelial electrical resistance (TEER). The expression of claudin-1, E-cadherin, N-cadherin, snail, slug, and activation of mitogen-activated proteins kinases (MAPKs) and AKT was assessed following TGF-β1 treatment using Western blot analysis. Claudin-1 expression was decreased in glandular BPH tissue compared with adjacent normal prostatic tissue in patient specimens. TGF-β1 treatment or claudin-1 knockdown in prostatic epithelial cell lines increased monolayer permeability. TGF-β1 decreased levels of claudin-1 and increased levels of snail and slug as well as increased phosphorylation of the MAPK extracellular signal-regulated kinase-1/2 (ERK-1/2) in both BPH-1 and BHPrE1 cells. Overexpression of snail or slug had no effect on claudin-1 expression. In contrast, PD98059 and U0126, inhibitors of the upstream activator of ERK-1/2 (ie, MEK-1/2) restored claudin-1 expression level as well as the epithelial barrier. Our findings suggest that downregulation of claudin-1 by TGF-β1 acting through the noncanonical MEK-1/2/ERK-1/2 pathway triggers increased prostatic epithelial monolayer permeability in vitro. These findings also suggest that elevated TGF-β1 may contribute to claudin-1 downregulation and compromised epithelial barrier in clinical BPH specimens.