Permeability Of Human Umbilical Vein Endothelial Cells Research Articles

Da-Cheng-Qi decoction improves severe acute pancreatitis capillary leakage syndrome by regulating tight junction-associated proteins.

To investigate mechanisms underlying the effects of Da-Cheng-Qi decoction (DCQD) on severe acute pancreatitis (SAP) capillary leakage syndrome. In this study, a SAP rat model was established using retrograde perfusion of 5% sodium taurocholate into the biliopancreatic duct. The study included three randomized groups: control, SAP (modeling), and DCQD (via gavage at 2h pre-modeling and 2 and 4h post-modeling). HPLC was used to analyzed major components of DCQD. Pathological changes and capillary permeability in the rat pancreatic tissues were examined. mRNA levels of claudin 5, occludin, zonula occludin-1 (ZO-1), and junctional adhesion molecules (JAM-C) were assessed using qRT-PCR. Tight junction-associated protein expression was evaluated using immunofluorescence and Western blot analyses. Human umbilical vein endothelial cells (HUVECs) were used to investigate the mechanism m of DCQD. Serum levels of amylase, TNF-α, IL-1β, IL-2, and IL-6 were higher in the SAP group compared to the DCQD group (p < 0.05). DCQD treatment significantly attenuated rat pancreas damage (p < 0.05) and reduced tissue capillary permeability compared to the SAP group (p < 0.05). Claudin 5, occludin, and ZO-1 expression in the rat tissues was upregulated, but JAM-C was downregulated by DCQD treatment (p < 0.05). HUVEC permeability was improved by DCQD in a dose-time-dependent manner compared to the SAP group (p < 0.05). DCQD also upregulated claudin 5, occludin, and ZO-1 expression in vitro (p < 0.05). DCQD can improve capillary permeability in both in vivo and in vitro models of SAP by upregulating expression of claudin 5, occludin, and ZO-1, but not JAM-C.

Macrophages-derived exo-miR-4449 induced by Cryptococcus affects HUVEC permeability and promotes pyroptosis in BEAS-2B via the HIC1 pathway

Macrophages have recently been discovered to assume a significant role in the progression of cryptococcosis. However, the potential involvement of macrophage-derived exosomes in the pathogenesis of cryptococcosis remains uncertain. In this study, we investigated the changes of microRNAs in macrophage exosomes (exo-miRNAs) in cryptococcal infections and the role of markedly altered exo-miRNAs in the modulation of Human Umbilical Vein Endothelial Cells (HUVEC) permeability and ROS accumulation and pyroptosis in Human Bronchial Epithelioid Cells (BEAS-2B). Techniques such as microarray analysis and real-time quantitative PCR were used to detect different exo-miRNAs and to screen for the most highly expressed exo-miRNAs. Then its mimics were transfected into HUVEC to study its effect on the monolayer permeability of HUVEC. Finally, the relationship between this exo-miRNAs and the ROS accumulation and pyroptosis was verified by bioinformatics analysis. The results showed that five exo-miRNAs were overexpressed and two exo-miRNAs were reduced, among which, exo-miR-4449 was expressed at the highest level. Exo-miR-4449 could be internalized by HUVEC and enhanced its monolayer permeability. Moreover, exo-miR-4449 was found to promote ROS accumulation and pyroptosis in BEAS-2B through HIC1 pathway. Thus, exo-miR-4449 plays an important role in the pathogenesis of cryptococcosis and holds promise as a significant biomarker for treatment.

Exosome-derived GTF2H2 from Huh7 cells can inhibit endothelial cell viability, migration, tube formation, and permeability

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related morbidity and mortality worldwide. Given that HCC is an extraordinarily heterogeneous malignant disease, finding an effective therapeutic strategy for treating it has been difficult. Because of the importance of angiogenesis in tumorigenesis, targeting the more homogenous HCC endothelial cells may be a better therapeutic strategy. In a unpublished manuscript, we found that the expression levels of vascular endothelial growth factor receptor 2 (VEGFR2) and matrix metalloproteinase 2/9 (MMP2/9) were reduced in human HCC tissues that overexpressed DNA damage repair gene general transcription factor II subunit H2 (GTF2H2). This suggested that GTF2H2 may have an inhibitory effect on angiogenesis. Therefore, we hypothesized that GTF2H2 acts as an anti-angiogenesis gene. However, our results showed that GTF2H2 overexpression had no effect on endothelial cell viability, migration, or permeability. To our surprise, treating human umbilical vein endothelial cells (HUVECs) with the culture medium of Huh 7 cells overexpressing GTF2H2 could inhibit their viability, migration, and permeability. We then isolated the culture medium into exosomes and other components from the culture medium. Only GTF2H2-enriched exosomes could inhibit the viability, migration, tube formation, and permeability of HUVECs. Our results suggest that overexpressing GTF2H2 had no effect on HUVECs, while GTF2H2 enriched exosomes from Huh7 cells could inhibit HUVEC phenotypes such as proliferation and migration. Therefore, GTF2H2-enriched exosomes can possibly be utilized as a novel drug for treating HCC and also serve as a potential molecular target for inhibiting tumor angiogenesis.

TCF7L2 promotes ER stress signaling in diabetic retinopathy

Diabetic retinopathy (DR) is one of the most common blindness in working-age adults. Transcription factor 7 like 2 (TCF7L2) is a susceptibility gene of DR, however, its roles in the pathogenesis of DR are still largely unknown. In this study, we found that TCF7L2 was mainly located in the cell nucleus of retinal ganglion cell layer (GCL) and inner nuclear layer (INL), while it was not expressed in the cell nucleus of retinal outer nuclear layer (ONL). Expression of TCF7L2 was significantly elevated in the retinas of db/db diabetic mice and oxygen-induced retinopathy (OIR) mice. Also, in Ad-hTCF7L2 treated hiPSCs-derived retinal progenitor cells (RPCs), activating transcription factor 6 (ATF6)-related endoplasmic reticulum (ER) stress signaling was remarkably activated. Moreover, knockdown of TCF7L2 significantly inhibited ATF6-related ER stress signaling. Furthermore, the data of endothelial permeability assay showed that RPCs pretreated with Ad-hTCF7L2 lead to enhanced monolayer permeability of human umbilical vein endothelial cells (HUVECs), and knockdown of TCF7L2 or ATF6 in RPCs could alleviate the monolayer permeability of HUVECs. Thus, our results showed that TCF7L2 could trigger ATF6-related ER stress signaling and promote vein endothelial cell permeability, which will provide important insight into the role of TCF7L2 in the pathogenesis of DR and contribute to designing potential therapies.

Somatostatin Primes Endothelial Cells for Agonist-Induced Hyperpermeability and Angiogenesis In Vitro

Somatostatin is an inhibitory peptide, which regulates the release of several hormones, and affects neurotransmission and cell proliferation via its five Gi protein-coupled receptors (SST1-5). Although its endocrine regulatory and anti-tumour effects have been thoroughly studied, little is known about its effect on the vascular system. The aim of the present study was to analyse the effects and potential mechanisms of somatostatin on endothelial barrier function. Cultured human umbilical vein endothelial cells (HUVECs) express mainly SST1 and SST5 receptors. Somatostatin did not affect the basal HUVEC permeability, but primed HUVEC monolayers for thrombin-induced hyperpermeability. Western blot data demonstrated that somatostatin activated the phosphoinositide 3-kinases (PI3K)/protein kinase B (Akt) and p42/44 mitogen-activated protein kinase (MAPK) pathways by phosphorylation. The HUVEC barrier destabilizing effects were abrogated by pre-treating HUVECs with mitogen-activated protein kinase kinase/extracellular signal regulated kinase (MEK/ERK), but not the Akt inhibitor. Moreover, somatostatin pre-treatment amplified vascular endothelial growth factor (VEGF)-induced angiogenesis (3D spheroid formation) in HUVECs. In conclusion, the data demonstrate that HUVECs under quiescence conditions express SST1 and SST5 receptors. Moreover, somatostatin primes HUVECs for thrombin-induced hyperpermeability mainly via the activation of MEK/ERK signalling and promotes HUVEC proliferation and angiogenesis in vitro.

Study on the Mechanism of MicroRNA551b-5p in Severe Acute Pancreatitis Capillary Leakage Syndrome.

Objective This study focused on investigating the effects of microRNA551b-5p (miR-551b-5p) on severe acute pancreatitis. Methods Initially, quantitative real-time polymerase chain reaction (qPCR) is employed to determine the expression of miR-551b-5p in differentiated human umbilical vein endothelial cells (HUVECs). Further, the effects of aberrantly expressed miR-551b-5p in HUVECs Transwell assay. The expressions of proteins associated with severe acute pancreatitis capillary leakage syndrome are determined by Western blot, FITC-phalloidin, and immunofluorescence stainings. Finally, the correlative factor and the target genes of miR-551b-5p, as well as their contributions, are assessed. Results We observed that overexpression of miR-551b-5p distinctly promoted the expression of EGFR, AKT3, and AQP5, while it suppressed the expression of JAM3, AQP1, and occludin. Functionally, the cytoskeleton of the miR-551b-5p overexpression was relatively loose with apparent vacuoles, and overexpression of miR-551b-5p increased the permeability of HUVECs. Conclusion miR-551b-5p overexpression promoted changes in vascular endothelial permeability via upregulation of the EGFR/AKT3 pathway and downregulation of occludin and JAM3.

Dabigatran: its protective effect against endothelial cell damage by oxysterol

Recent data showed that dabigatran can reduce not only procoagulatory effects but also block proinflammatory stimuli by inhibiting the expression of cytokines and chemokines and reducing thrombin-induced endothelial permeability. The aim of our study was to assess the effect of dabigatran on the integrity and inflammatory properties of endothelial cells stimulated by 25-hydroxycholesterol (25-OHC, oxysterol). HUVECs (Human Umbilical Vein Endothelial Cells) were stimulated with 25-hydroxycholesterol 10 µg/ml, dabigatran 100 ng/ml or 500 ng/ml and 25-hydroxycholesterol + dabigatran (100 ng/ml, 500 ng/ml). HUVEC integrity and permeability was measured in the RTCA-DP xCELLigence system and by the paracellular flux system. The mRNA expression of ICAM-1, VEGF, IL-33, MCP-1 and TNF-α was analyzed by Real-time PCR. Cell apoptosis and viability was measured by flow cytometry. VEGF protein concentration was assessed in supernatants by ELISA. VE-cadherin expression in endothelial cells was evaluated by confocal microscopy. Pre-stimulation of HUVECs with 25-OHC decreased endothelial cell integrity (p < 0.001) and increased the expression of IL-33, ICAM-1, MCP-1, VEGF, TNF-α mRNA (p < 0.01) compared to unstimulated controls. Following stimulation of HUVECs with dabigatran 100 ng/ml or 500 ng/ml restored HUVEC integrity interrupted by 25-OHC (p < 0.001). In HUVECs pre-stimulated with oxysterol, dabigatran stimulation decreased mRNA expression of the proinflammatory cytokines IL-33 and TNF-α, chemokines MCP-1 ICAM-1 and VEGF (p < 0.01). Dabigatran 500 mg/ml+ 25-OHC increased the endothelial expression of VE-cadherin as compared to 25-OHC (p < 0.01).Our findings suggest that dabigatran stabilizes the endothelial barrier and inhibits the inflammation caused by oxysterol.

The C Loop of Fifth Epidermal Growth Factor-like Domain of Thrombomodulin Exerts Cytoprotective and Angiogenic Effects in Vitro and In Vivo

We previously found that the fifth epidermal growth factor-like domain of thrombomodulin (TME5) exerted cytoprotective and pro-angiogenic function in vitro and in vivo via G-protein coupled receptor 15 (GPR15). TME5 comprises three S-S bonds which divides TME5 into three loops: A (TME5A), B (TME5B) and C (TME5C). The present study aimed to identify the minimum structure of TME5 that produces favorable effects in vascular endothelial cells. The bromodeoxyuridine (BrdU) incorporation and in vivo Matrigel plug assays found that TME5C but not TME5A or TME5B stimulated proliferation of human umbilical vein endothelial cells (HUVECs) and in vivo angiogenesis, respectively. In addition, TME5C counteracted calcineurin inhibitor-induced apoptosis and vascular permeability in HUVECs. Western blot analysis found that exposure of HUVECs to TME5C increased levels of anti-apoptotic myeloid cell leukemia-1 (Mcl-1) protein in association with activation of signal transduction pathways including extracellular signal-regulated kinase (ERK), AKT and mitogen-activated protein kinase p38. Importantly, TME5C did not affect thrombin clotting time in vitro. The cytoprotective function of TME5C was mediated by cell surface expressed GPR15, as TME5C was not able to protect vascular endothelial cells isolated from GPR15 knockout mice. Strikingly, TME5C successfully ameliorated hematopoietic stem cell transplantation (HSCT)-induced sinusoidal obstruction syndrome (SOS) in a murine model in association with counteracting reduction of sinusoidal ECs numbers, loss of integrity of sinusoidal wall and sinusoidal ECs detachment. Taken together, cytoprotective and the pro-angiogenetic functions of TM are preserved in TME5C. Use of TME5C may be a promising treatment strategy to prevent or treat lethal complications such as SOS whose pathogenesis is based on the endothelial insults. DisclosuresNo relevant conflicts of interest to declare.

Exosomal miR-22-3p Derived from Chronic Rhinosinusitis with Nasal Polyps Regulates Vascular Permeability by Targeting VE-Cadherin.

Background The abnormal vascular permeability is associated with the formation of chronic rhinosinusitis with nasal polyps (CRSwNP). Previously, our study demonstrated that the nasal lavage fluid- (NLF-) derived exosomes from CRSwNP can promote the vascular permeability of human umbilical vein endothelial cells (HUVECs). miR-22-3p, a specific differentiated miRNA, is reported to regulate microvessels in some diseases. This study is purposed to explore the impact of exosomal miR-22-3p derived from CRSwNP on vascular permeability and identify the underlying targets. Methods Exosomes were extracted from NLF of 26 CRSwNP patients and 10 control patients. Quantitative real-time PCR (qRT- PCR) was applied to evaluate the relative level of exosomal miR-22-3p. The impact of exosomal miR-22-3p on HUVECs was assessed by permeability assays in vitro. The potential molecular targets of miR-22-3p were investigated by applying such technologies as dual-luciferase reporter assay and western blot. Results miR-22-3p was upregulated in NLF-derived exosomes from CRSwNP. Exosomal miR-22-3p derived from CRSwNP enhanced the tubule permeability of HUVECs. Vascular endothelial- (VE-) cadherin (CDH5) was identified as a direct target of miR-22-3p. miR-22-3p regulated the vascular permeability by targeting VE-cadherin in HUVECs. Conclusions Exosomal miR-22-3p derived from NLF of CRSwNP plays an important role in regulating vascular permeability by targeting VE-cadherin.

HnRNPA2/B1 Ameliorates LPS-Induced Endothelial Injury through NF-κB Pathway and VE-Cadherin/β-Catenin Signaling Modulation In Vitro.

Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) is a protein involved in the regulation of RNA processing, cell metabolism, migration, proliferation, and apoptosis. However, the effect of hnRNPA2/B1 on injured endothelial cells (ECs) remains unclear. We investigated the effect of hnRNPA2/B1 on lipopolysaccharide- (LPS-) induced vascular endothelial injury in human umbilical vein endothelial cells (HUVECs) and the underlying mechanisms. LPS was used to induce EC injury, and the roles of hnRNPA2/B1 in EC barrier dysfunction and inflammatory responses were measured by testing endothelial permeability and the expression of inflammatory factors after the suppression and overexpression of hnRNPA2/B1. To explore the underlying mechanism by which hnRNPA2/B1 regulates endothelial injury, we studied the VE-cadherin/β-catenin pathway and NF-κB activation in HUVECs. The results showed that hnRNPA2/B1 was elevated in LPS-stimulated HUVECs. Moreover, knockdown of hnRNPA2/B1 aggravated endothelial injury by increasing EC permeability and promoting the secretion of the inflammatory cytokines TNF-α, IL-1β, and IL-6. Overexpression of hnRNPA2/B1 can reduce the permeability and inflammatory response of HUVEC stimulated by LPS in vitro, while increasing the expression of VE-Cadherin and β-catenin. Furthermore, the suppression of hnRNPA2/B1 increased the LPS-induced NF-κB activation and reduced the VE-cadherin/β-catenin pathway. Taken together, these results suggest that hnRNPA2/B1 can regulate LPS-induced EC damage through regulating the NF-κB and VE-cadherin/β-catenin pathways.

Inhibition of Protein Prenylation of GTPases Alters Endothelial Barrier Function.

The members of Rho family of GTPases, RhoA and Rac1 regulate endothelial cytoskeleton dynamics and hence barrier integrity. The spatial activities of these GTPases are regulated by post-translational prenylation. In the present study, we investigated the effect of prenylation inhibition on the endothelial cytoskeleton and barrier properties. The study was carried out in human umbilical vein endothelial cells (HUVEC) and protein prenylation is manipulated with various pharmacological inhibitors. Inhibition of either complete prenylation using statins or specifically geranylgeranylation but not farnesylation has a biphasic effect on HUVEC cytoskeleton and permeability. Short-term treatment inhibits the spatial activity of RhoA/Rho kinase (Rock) to actin cytoskeleton resulting in adherens junctions (AJ) stabilization and ameliorates thrombin-induced barrier disruption whereas long-term inhibition results in collapse of endothelial cytoskeleton leading to increased basal permeability. These effects are reversed by supplementing the cells with geranylgeranyl but not farnesyl pyrophosphate. Moreover, long-term inhibition of protein prenylation results in basal hyper activation of RhoA/Rock signaling that is antagonized by a specific Rock inhibitor or an activation of cAMP signaling. In conclusion, inhibition of geranylgeranylation in endothelial cells (ECs) exerts biphasic effect on endothelial barrier properties. Short-term inhibition stabilizes AJs and hence barrier function whereas long-term treatment results in disruption of barrier properties.

Malaysian Tualang Honey Inhibits Hydrogen Peroxide-Induced Endothelial Hyperpermeability.

Malaysian Tualang honey (TH) is a known therapeutic honey extracted from the honeycombs of the Tualang tree (Koompassia excelsa) and has been reported for its antioxidant, anti-inflammatory, antiproliferative, and wound healing properties. However, the possible vascular protective effect of TH against oxidative stress remains unclear. In this study, the effects of TH on hydrogen peroxide- (H2O2-) elicited vascular hyperpermeability in human umbilical vein endothelial cells (HUVECs) and Balb/c mice were evaluated. Our data showed that TH concentrations ranging from 0.01% to 1.00% showed no cytotoxic effect to HUVECs. Induction with 0.5 mM H2O2 was found to increase HUVEC permeability, but the effect was significantly reversed attenuated by TH (p < 0.05), of which the permeability with the highest inhibition peaked at 0.1%. In Balb/c mice, TH (0.5 g/kg-1.5 g/kg) significantly (p < 0.05) reduced H2O2 (0.3%)-induced albumin-bound Evans blue leak, in a dose-dependent manner. Immunofluorescence staining confirmed that TH reduced actin stress fiber formation while increasing cortical actin formation and colocalization of caveolin-1 and β-catenin in HUVECs. Signaling studies showed that HUVECs pretreated with TH significantly (p < 0.05) decreased intracellular calcium release, while sustaining the level of cAMP when challenged with H2O2. These results suggested that TH could inhibit H2O2-induced vascular hyperpermeability in vitro and in vivo by suppression of adherence junction protein redistribution via calcium and cAMP, which could have a therapeutic potential for diseases related to the increase of both oxidant and vascular permeability.

Effect of melatonin on EGF- and VEGF-induced monolayer permeability of HUVECs.

Melatonin is a natural hormone involved in the regulation of circadian rhythm, immunity, and cardiovascular function. In the present study, we focused on the mechanism of melatonin in the regulation of vascular permeability. We found that melatonin could inhibit both VEGF- and EGF-induced monolayer permeability of human umbilical vein endothelial cells (HUVECs) and change the tyrosine phosphorylation of vascular-endothelial (VE-)cadherin, which was related to endothelial barrier function. In addition, phospho-AKT (Ser473) and phospho-ERK(1/2) played significant roles in the regulation of VE-cadherin phosphorylation. Both the phosphatidylinositol 3-kinase/AKT inhibitor LY49002 and MEK/ERK inhibitor U0126 could inhibit the permeability of HUVECs, but with different effects on tyrosine phosphorylation of VE-cadherin. Melatonin can influence the two growth factor-induced phosphorylation of AKT (Ser473) but not ERK(1/2). Our results show that melatonin can inhibit growth factor-induced monolayer permeability of HUVECs by influencing the phosphorylation of AKT and VE-cadherin. Melatonin can be a potential treatment for diseases associated with abnormal vascular permeability. NEW & NOTEWORTHY We found that melatonin could inhibit both EGF- and VEGF-induced monolayer permeability of human umbilical vein endothelial cells, which is related to phosphorylation of vascular-endothelial cadherin. Blockade of phosphatidylinositol 3-kinase/AKT and MEK/ERK pathways could inhibit the permeability of human umbilical vein endothelial cells, and phosphorylation of AKT (Ser473) might be a critical event in the changing of monolayer permeability and likely has cross-talk with the MEK/ERK pathway.

Cytoprotective and pro-angiogenic functions of thrombomodulin are preserved in the C loop of the fifth epidermal growth factor-like domain.

We previously found that the fifth epidermal growth factor-like domain of thrombomodulin (TME5) exerts cytoprotective and pro-angiogenic functions via G-protein coupled receptor 15 (GPR15). TME5 is comprised of three S-S bonds that divide it into three loops: A (TME5A), B (TME5B), and C (TME5C). Herein we identified the minimum structure of TME5 that produces favorable effects in vascular endothelial cells (ECs). We found that TME5C, composed of 19 amino acids, but not TME5A or TME5B, stimulated the proliferation of human umbilical vein endothelial cells (HUVECs) and human hepatic sinusoidal endothelial cells (HHSECs). Matrigel plug assays showed that TME5C stimulates in vivo angiogenesis. In addition, TME5C counteracted calcineurin inhibitor-induced apoptosis and vascular permeability in HUVECs and HHSECs. Western blot analysis indicated that exposure of either HUVECs or HHSECs to TME5C increased the levels of anti-apoptotic myeloid cell leukemia-1 protein in association with the activation of signal transduction pathways, including extracellular signal-regulated kinase, AKT, and mitogen-activated protein kinase p38. Importantly, TME5C did not affect the coagulation pathway in vitro. The cytoprotective function of TME5C was mediated by cell surface-expressed GPR15, as TME5C was not able to protect vascular ECs isolated from Gpr15 knock-out (KO) mice. Strikingly, TME5C successfully ameliorated sinusoidal obstruction syndrome in a murine model by counteracting the reduction of sinusoidal EC numbers. Taken together, the cytoprotective and pro-angiogenetic functions of TM are preserved in TME5C. The use of TME5C may be a promising treatment strategy to prevent or treat lethal complications, such as sinusoidal obstruction syndrome, whose pathogenesis is based on endothelial insults.

A disintegrin and metalloprotease 10-containing exosomes derived from nasal polyps promote angiogenesis and vascular permeability.

Abnormal angiogenesis and vascular permeability is important for the formation of nasal polyps (NPs). Increasing evidence has indicated that exosomes serve a vital role in modulating angiogenesis and vascular permeability. A disintegrin and metalloprotease 10 (ADAM10), an important type of proteinase that is overexpressed in various diseases, can influence angiogenesis and vascular permeability and has been observed in healthy nasal exosomes. To the best of our knowledge, the expression levels and the function of ADAM10 in NLF-derived exosomes from NPs has not been demonstrated previously. In order to determine the influence of exosomes derived from nasal lavage fluid (NLF) on angiogenesis and vascular permeability, 25 nasal polyp patients and 15 healthy volunteers were enrolled in the present study. NLF was collected from all of the subjects. Exosomes were isolated from NLF, visualized under transmission electron microscope and identified using western blot analysis. The effect of exosomes on human umbilical vein endothelial cells (HUVECs) was measured by tube formation and permeability assays in vitro. The expression of exosomal ADAM10 was also analyzed by western blotting. NLF-derived exosomes from NPs influenced proliferation, tube formation and the permeability of HUVECs. ADAM10 was highly expressed in NLF-derived exosomes from NPs when compared with healthy volunteers. Thus, NLF-derived exosomes from NPs promoted angiogenesis and vascular permeability, which may be associated with abundant ADAM10 in NP exosomes.

Formyl peptide receptor-2 is decreased in foetal growth restriction and contributes to placental dysfunction.

What is the association between placental formyl peptide receptor 2 (FPR2) and trophoblast and endothelial functions in pregnancies affected by foetal growth restriction (FGR)? Reduced FPR2 placental expression in idiopathic FGR results in significantly altered trophoblast differentiation and endothelial function in vitro. FGR is associated with placental insufficiency, where defective trophoblast and endothelial functions contribute to reduced feto-placental growth. The expression of FPR2 in placental tissues from human pregnancies complicated with FGR was compared to that in gestation-matched uncomplicated control pregnancies (n = 25 from each group). Fpr2 expression was also determined in placental tissues obtained from a murine model of FGR (n = 4). The functional role of FPR2 in primary trophoblasts and endothelial cells in vitro was assessed in diverse assays in a time-dependent manner. Placentae from third-trimester pregnancies complicated by idiopathic FGR (n = 25) and those from gestation-matched pregnancies with appropriately grown infants as controls (n = 25) were collected at gestation 27-40 weeks. Placental tissues were also collected from a spontaneous CBA/CaH × DBA/2 J murine model of FGR. Placental FPR2/Fpr2 mRNA expression was determined by real-time PCR, while protein expression was examined by immunoblotting and immunohistochemistry. siRNA transfection was used to silence FPR2 expression in primary trophoblasts and in human umbilical vein endothelial cells (HUVEC), and the quantitation of cytokines, chemokines and apoptosis was performed following a cDNA array analyses. Functional effects of trophoblast differentiation were measured using HCGB/β-hCG and syncytin-2 expression as well as markers of apoptosis, tumour protein 53 (TP53), caspase 8, B cell lymphoma 2 (BCL2) and BCL associated X (BAX). Endothelial function was assessed by proliferation, network formation and permeability assays. Placental FPR2/Fpr2 expression was significantly decreased in FGR placentae (n = 25, P < 0.05) as well as in murine FGR placentae compared to controls (n = 4, P < 0.05). FPR2 siRNA (siFPR2) in term trophoblasts significantly increased differentiation markers, HCGB and syncytin-2; cytokines, interleukin (IL)-6, CXCL8; and apoptotic markers, TP53, caspase 8 and BAX, but significantly reduced the expression of the chemokines CXCL12 and its receptors CXCR4 and CXCR7; CXCL16 and its receptor, CXCR6; and cytokine, IL-10, compared with control siRNA (siCONT). Treatment of HUVECs with siFPR2 significantly reduced proliferation and endothelial tube formation, but significantly increased permeability of HUVECs. N/A. Reduced expression of placental FPR2/Fpr2 was observed in the third trimester at delivery after development of FGR, suggesting that FPR2 is associated with FGR pregnancies. However, there is a possibility that the decreased placental FPR2 observed in FGR may be a consequence rather than a cause of FGR, although our in vitro functional analyses using primary trophoblasts and endothelial cells suggest that FPR2 may have a direct or indirect regulatory role on trophoblast differentiation and endothelial function in FGR. This is the first study linking placental FPR2 expression with changes in the trophoblast and endothelial functions that may explain the placental insufficiency observed in FGR. P.M. and P.R.E. received funding from the Australian Institute of Musculoskeletal Science, Western Health, St. Albans, Victoria 3021, Australia. M.L. is supported by a Career Development Fellowship from the National Health and Medical Research Council (NHMRC; Grant no. 1047025). Monash Health is supported by the Victorian Government's Operational Infrastructure Support Programme. The authors declare that there is no conflict of interest in publishing this work.

Effects of oxygen-glucose deprivation on the permeability of human umbilical vein endothelial cells

To investigate the effects of oxygen-glucose deprivation (OGD) on the functions of blood-brain barrier in vitro, we established an OGD model to mimic cerebral ischemic injury in human umbilical vein endothelial cells (HUVECs). Cells were cul-tured in DMEM without glucose under hypoxic conditions (0.3% O2) for different time point. The survival rate of HUVECs was detected by CCK-8 assay. The permeability of HUVECs was measured by epithelial volt-ohm meter. The expression of tight junction protein was detected by Western blot analysis. In OGD group, the survival rate of HUVECs was obviously decreased. The permeability of HUVECs also decreased in OGD-treated cells. And the expression of tight junction protein was reduced after OGD treatment. Our data indicate that OGD treatment disrupt the barrier function of tight junctions and increase permeability of HUVECs and finally accelerated cell death.

Exposure to endosulfan increases endothelial permeability by transcellular and paracellular pathways in relation to cardiovascular diseases

Exposure to environmental pollutants results in out-of-balance of vascular homeostasis. Endothelial dysfunction leads to a disruption of the endothelial permeability characteristics, associated with cardiovascular diseases. We previously reported that endosulfan could cause endothelial dysfunction, but the role of endosulfan in permeability of endothelial cells has been unexplored. To elucidate molecular mechanism of endosulfan-induced changes in endothelial permeability, human umbilical vein endothelial cells (HUVECs) were exposed to endosulfan, followed by endothelial permeability analysis. The results showed that permeability of HUVECs was enhanced at 48 h after exposure to endosulfan in a dose-dependent manner. Immunofluorescence analysis demonstrated the disruptions of actin cytoskeleton and focal adhesion in endosulfan-exposed cells. Endosulfan activated MMP3/LAMC1/FAK signaling pathway, and downregulated ROCK and PXN in transcellular pathway. Endosulfan affected adherens junctions via E-cadherin and β-catenin, and impaired gap junctions through downregulation of Cx43 in paracellular pathway. We predicted four closely related human cardiovascular diseases in Nextbio, including shock, coronary arteriosclerosis, disorder of cardiac function and hypertensive disorder in relation to endosulfan exposure. Some genes such as ROCK2 and PXN were predicted to be key genes in these diseases. These findings suggest that endosulfan increased endothelial permeability by paracellular and transcellular pathways, implicating the potential correlation between endosulfan and cardiovascular diseases.

Dachengqi Decoction Attenuates Intestinal Vascular Endothelial Injury in Severe Acute Pancreatitis in Vitro and in Vivo

Background/Aims: Dachengqi decoction (DCQD) is a well-known traditional Chinese herbal drug with strong anti-inflammatory effects. Angiopoietin-1 (Ang-1) plays a vital role in maintaining the stability and integrity of the vascular wall and prevents vascular leakage due to inflammatory mediators. Our previous work found that DCQD protects against pancreatic injury in rats with severe acute pancreatitis (SAP). This study aims to investigate the effects of DCQD on intestinal endothelial damage in both damaged human umbilical vein endothelial cells (HUVECs) and SAP rats. Methods: HUVECs were randomly divided into four groups: control group, TNF-α group, TNF-α plus Ang-1 group (Ang-1 group), and TNF-α plus DCQD group (DCQD group). Cells were incubated for 6 h, 12 h, and 24 h, before collection. The treatment concentration of DCQD was decided based on a Cell Counting Kit-8 (CCK-8) assay. The monolayer permeability of the HUVECs was assessed by measuring the transendothelial electrical resistance (TEER). Apoptosis was analyzed by flow cytometry. mRNA and protein expression of aquaporin 1 (AQP-1), matrix metalloproteinase 9 (MMP9), and junctional adhesion molecule-C (JAM-C) was evaluated by RT-PCR, immunocytofluorescence, and western blot. Forty male Sprague-Dawley rats were randomized into a control group, SAP group, SAP plus Ang-1 group (Ang-1 group), and SAP plus DCQD group (DCQD group). SAP was induced by intraperitoneal injection of cerulein and lipopolysaccharide (LPS), while the control group received 0.9% saline solution. Evans blue was injected through the penile vein and the rats were then sacrificed 12 h after modeling. Levels of serum amylase, TNF-α, IL-1β, IL-2, and IL-6 were determined by using ELISA. Intestinal tissue was analysed by histology, and capillary permeability in the tissues was evaluated by Evans blue extravasation assay. Protein and mRNA expression of AQP-1, MMP9, and JAM-C were assessed by immunohistofluorescence, western blot, and RT-PCR. Results: DCQD reduced the permeability of HUVEC induced by TNF-α in vitro. Furthermore, DCQD altered the mRNA and protein levels of JAM-C, MMP9, and AQP-1 in HUVECs after TNF-α induction. SAP intestinal injury induced by cerulein combined with lipopolysaccharides was concomitant with increased expression of JAM-C and MMP9, and reduced expression of AQP-1 in intestinal tissue. Pretreatment with DCQD attenuated SAP intestinal injury and lowered the levels of serum amylase, TNF–α, IL-1β, IL-2, and IL-6 effectively. Our study demonstrated that DCQD decreased the expression of JAM-C and MMP9 and increased the expression of AQP-1 both in vitro and in vivo. Conclusion: DCQD can reduce capillary endothelial damage in acute pancreatitis-associated intestinal injury and the mechanism may be associated with the regulation of endothelial barrier function-associated proteins AQP-1, MMP9, and JAM-C.

Endothelial JAK2V617F Expression Drives Inflammation and Cellular Senescence; New Evidence for the Roles of Endothelial Cells in MPN-Related Clotting Abnormalities?

JAK2V617F is the primary pathogenic mutation in patients with Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), a heterogeneous group of haematological malignancies characterised by increases in one or more myeloid lineages. The most common cause of morbidity and mortality in patients with MPNs are thrombohemorrhagic incidents, although the events leading to these clotting abnormalities remain unclear. It is now known that a subset of MPN patients express JAK2V617F in endothelial cells (ECs) which has been linked with a poorer prognosis and an increase in the incidence of thrombosis. Furthermore, using transgenic mouse models we have recently shown that expression of JAK2V617F in ECs plays a critical role in aberrant thrombosis in vivo (PNAS, 2014. 111(6): p. 2295-300). Here, we describe the transcriptomic and phenotypic profiles of JAK2V617F-positive ECs.Lentivirally transduced human umbilical vein ECs (HUVECs) showed a sustained increase in levels of activated JAK2, STAT3 and STAT1 in JAK2V617F-expressing cells compared to WT JAK2. In addition, JAK2V617F also caused a significant increase in expression of total STAT1 protein. Subsequent RNAseq analysis revealed an upregulation of several mRNAs related to inflammation and permeability in JAK2V617F-expressing cells. Expression of interferon-related genes, including serine protease inhibitor B2 (SERPINB2) and early growth response protein 1 (EGR1), and chemokine ligand 2 (CCL2) were also increased in JAK2V617F cells. SERPINB2 and EGR1 have roles in initiating cellular senescence and CCL2 was previously reported to induce permeability. These data, combined with increased expression of total STAT1, suggest that JAK2V617F expression in ECs induces an inflammatory response in this cell type. Moreover, in vitro permeability assays show increased permeability in HUVECs transduced with JAK2V617Fcompared to WT JAK2, which was significantly amplified following stimulation with thrombin.We then determined whether any functional differences existed between patient-derived normal and JAK2V617F-expressing ECs. For this we obtained blood from JAK2V617F-positive polycythaemia vera patients undergoing therapeutic phlebotomy and isolated blood outgrowth endothelial cells (BOECs) by repeated culture of PBMCs. Allelic burden of JAK2V617F, determined by quantitative allele-specific amplification (QuASA) was shown to vary between patient samples, (0% to 59.8% (n=9)) and increased expression of JAK2V617F appeared to correlate with decreased proliferation and significant changes in cellular morphology.In order to quantify these differences, ptychography, a novel label-free imaging technology was used to generate quantitative phase images of BOECs in time-lapse, to measure phenotypic changes in cell morphology, motility and proliferation. These results confirmed that JAK2V617F allelic burden was inversely related to BOEC proliferation. Cells which expressed only WT JAK2 or low levels of JAK2V617F exhibited exponential growth. However, an increase in JAK2V617F resulted in a decrease in BOEC proliferation. Indeed, cells which expressed high JAK2V617F levels (30-60%) failed to proliferate over the 48 hour imaging period. Furthermore, JAK2V617F-high BOECs were also shown to be larger in size, multi-nucleated and less motile compared to JAK2V617F-low. The increase in cell size and decreases in cell motility and proliferation suggest that increased JAK2V617F expression induces senescence in BOECs.These data describe features of ECs which are commonly associated with dysfunctional endothelium: sustained inflammation/permeability, limited cell growth and senescence. The critical role of ECs in maintaining hemostasis and the observed features of EC dysfunction in cells which express JAK2V617F, implicate mutation burden in ECs as a contributing factor towards the thrombotic abnormalities observed in patients with MPNs. DisclosuresNo relevant conflicts of interest to declare.