Human Microvascular Endothelial Cell Line Research Articles

Propofol attenuated TNF-\u03b1-modulated occludin expression by inhibiting Hif-1\u03b1/ VEGF/ VEGFR-2/ ERK signaling pathway in hCMEC/D3 cells

BackgroundThe levels of tight junction proteins (TJs), especially occludin, correlate with blood-brain barrier (BBB) disruption caused by inflammation in central nervous system (CNS). It has been reported that propofol, the most commonly used anesthetic, could inhibit inflammation response in CNS. In this study, we investigated the effects of tumor necrosis factor-α (TNF-α) and propofol on occludin expression in human cerebral microvascular endothelial cell line, D3 clone (hCMEC/D3 cells), and explored the underlying mechanisms.MethodsThe hCMEC/D3 cells were treated with propofol, followed by TNF-α. The expression and phosphorylation of Hif-1α, VEGF, VEGFR-2, ERK, p38MAPK and occludin were measured by Western blot analysis. The cell viability of hCMEC/D3 cells was measured by cell counting kit-8.ResultsTNF-α (10 ng/ml, 4 h) significantly decreased the expression of occludin, which was attenuated by propofol (25 μM). TNF-α induced Hif-1α/VEGF/VEGFR-2/ERK signaling pathway, while propofol could inhibit it. TNF-α induced the phosphorylation of p38MAPK, while propofol had no effect on it. In addition, the inhibitors of Hif-1α, VEGFR-2, and ERK could reduce the effect of TNF-α on occludin expression.ConclusionTNF-α could decrease the expression of occludin via activating Hif-1α/ VEGF/ VEGFR-2/ ERK signaling pathway, which was attenuated by propofol.

Amyloid beta25-35 impairs docosahexaenoic acid efflux by down-regulating fatty acid transport protein 1 (FATP1/SLC27A1) protein expression in human brain capillary endothelial cells.

Decreased levels of docosahexaenoic acid (DHA), an endogenous neuroprotective compound, in the brain are associated with the development of Alzheimer's disease (AD). We previously showed that DHA is a substrate of fatty acid transport protein 1 (FATP1/SLC27A1), and FATP1 is localized at the abluminal membrane of brain capillary endothelial cells. We hypothesized that amyloid β (Aβ) decreases FATP1-mediated cellular efflux (i.e. supply to the brain) of DHA at the blood-brain barrier (BBB). Here, we tested this hypothesis using a human cerebral microvascular endothelial cell line, human cerebral microvessel endothelial cells (hCMEC/D3), as a BBB model. The efflux of DHA-d5 by hCMEC/D3 cells increased time-dependently up to 3min. Knock-down of FATP1 with specific siRNA indicated that FATP1-mediated efflux accounts for 47.0% of this DHA-d5 efflux. In hCMEC/D3 cells treated with Aβ25-35 (10μM/24h), which we employed as an in vitro model of the BBB in AD, FATP1 protein expression in the plasma membrane was decreased by 96.0%, which was greater than the decrease in the whole-cell lysate, and the DHA-d5 efflux was decreased by 68.3%. Of this 68.3% decrease, 45.1% (47.0×0.96) is accounted for by the decrease in FATP1-mediated efflux and the remaining 23.2% is presumably mediated by other mechanism(s). Thus, we have established for the first time that FATP1 is a major contributor to DHA efflux from human brain capillary endothelial cells, and its efflux activity at the abluminal membrane of the cells is blocked by Aβ. This may explain the decreased DHA level in the brain of AD patients. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

A multiparametric study of gold nanoparticles cytotoxicity, internalization and permeability using an in vitro model of blood–brain barrier. Influence of size, shape and capping agent

Gold nanoparticles (AuNPs) have biomedical application on imaging and due to increased optical performance, star-shaped AuNPs are of special interest. Because shape, size and capping greatly influence their toxicokinetics and toxicodynamics, a systematic multiparametric comparative study of the influence of these parameters on the cytotoxicity, internalization, and in vitro permeability was conducted in human Cerebral Microvascular Endothelial Cell line (hCMEC/D3), an in vitro model of the human blood-brain barrier (BBB). AuNPs of different size (14 nm and ∼50 nm), shape (spheres and stars), and coating (11-mercaptoundecanoic acid or MUA and sodium citrate) were synthesized and fully characterized. The time- and concentration-dependent cytotoxic profile of the tested AuNPs differed for the different AuNPs. Generally, toxicity was greater for stars relative to sphere-shaped AuNPs, and citrate coating was more toxic than MUA. Regarding the influence of size, smaller-sized AuNPs were more cytotoxic when compared at the same Au concentration. However, when the concentration of AuNPs was expressed as the number of AuNPs/mL, a higher degree of cytotoxicity was noted for the larger ̴50 nm AuNPs. To understand the influence of size, shape and capping, a systematic study design, in which only one of the variables changes, is determinant for correct data interpretation. Considering the results herein presented, for the sake of comparison of differently-sized AuNPs, it is preferable to design the study based upon the number of nanoparticles, since at a fixed Au concentration the number of particles available to promote effect is higher for smaller-sized AuNPs. Cellular internalization also differed among the tested AuNPs; although all were unable to cross the in vitro BBB, the intracellularly accumulated AuNPs can induce cell damage and later compromise BBB integrity and permeability.

Reelin Signaling in Vascular Endothelial Cell Biology

IntroductionEstablishment of the coronary circulation is a critical step in heart development. Formation of functional coronary vessels is required for the heart to undergo successful compaction of the ventricular myocardium. Oxygen and nutrients are delivered to the contractile myocardium through the coronary vasculature. Molecular signaling pathways that control formation of the coronary vessels are being identified. Immunofluorescent studies from our lab indicate that the Reelin extracellular matrix glycoprotein is expressed within the vascular endothelial cells of developing and mature coronary vessels of the mouse heart. The Reelin signaling pathway is known to contribute to brain and lymphatic system development by influencing neuronal and endothelial cell behaviors such as adhesion and migration. However, its role in coronary vascular development is as yet unidentified.Study ObjectiveThe goal of this study was to elucidate the potential roles of Reelin in formation of the coronary vasculature by identifying how it regulates vascular endothelial cell behaviors. We hypothesized that Reelin signaling regulates establishment of coronary vessel integrity during cardiogenesis by influencing vascular endothelial cell adhesion as well as cell membrane permeability and resistance.MethodsWe utilized the primary human dermal microvascular endothelial cell line (HDMEC) as an in vitro system to examine contributions of Reelin to vascular endothelial cell behavior. A small interfering RNA‐mediated approach was used to silence the endogenous RELN gene in HDMECs and reduce Reelin protein expression. Transfected HDMECs were subjected to cell‐based assays to assess changes in vascular endothelial cell behavior in response to RELN silencing. We assessed vascular endothelial cell adhesion on extracellular matrices that are required during coronary vessel development. Vessel integrity was examined by assaying endothelial cell membrane permeability and resistance in RELN‐silenced cells. Also, we examined the effect of Reelin expression on cell viability.ResultsRELN‐silenced HDMECs exhibited a >90% reduction in Reelin expression in comparison to non‐targeting, negative control HDMECs. This significant Reelin reduction altered vascular endothelial cell adhesion to certain extracellular matrices. These RELN‐silenced HDMECs exhibited a significant decrease in baseline cell membrane permeability and concomitant changes in cell membrane resistance. Furthermore, we observed changes in cell morphology in RELN‐silenced cells versus control cells. Preliminary data suggests that loss of RELN expression affects vascular endothelial cell morphology and expression of components within the Reelin signaling pathway.ConclusionsBased on this evidence, we conclude that Reelin influences adhesion, morphology, membrane permeability and membrane resistance of vascular endothelial cells in order to regulate vessel wall integrity. These findings may provide key evidence as to how Reelin expression in nascent vascular endothelial cells impacts formation of the coronary vessels during mammalian cardiovascular development.Support or Funding InformationAHA 17AIREA3360773 and PCOM Center for Chronic Disorders of AgingThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Omi/HtrA2 Protease Associated Cell Apoptosis Participates in Blood-Brain Barrier Dysfunction.

Background: Omi/HtrA2 is a proapoptotic mitochondrial serine protease involved in caspase-dependent cell apoptosis, translocating from mitochondria to the cytosol after an apoptotic insult. Our previous study indicated pre-treatment with UCF-101, a specific inhibitor of Omi/HtrA2, could significantly reduce neuronal apoptosis and attenuate sepsis-induced cognitive dysfunction. Various hypotheses involving blood-brain-barrier (BBB) disruption have been proposed to account for sepsis-associated encephalopathy (SAE). Here, we attempted to explore whether interference of Omi/HtrA2 by RNA interference or UCF-101 pre-treatment can improve sepsis-induced disruption of BBB using human cerebral microvascular endothelial cell line (hCMEC/D3) in vitro and if so, to explore mechanisms involved Omi/HtrA2 protease mediates BBB disruption in SAE.Methods: hCMEC/D3 cell monolayers were intervened by different concentrations of LPS (0–50 μg/mL) over experimental period. Pharmacological or gene interventions (by silencing RNA of Omi/HtrA2) were used to study molecular mechanisms involved in sepsis-associated Omi/HtrA2 translocation, cell apoptosis and BBB dysfunction. BBB function was assessed by trans-endothelial electrical resistance (TEER) and permeability to labeled dextrans (FITC-4kDa). Tight junction (TJ) integrity was assessed by immunofluorescence, western blotting and transmission electron microscopic (TEM) analyses. Apoptosis was determined using flow cytometry and TUNEL assay. Mitochondrial membrane potential (MMP) and oxidative stress were also investigated.Results: LPS affects hCMEC/D3 TJ permeability in a concentration- and time-dependent manner. LPS intervention resulted in a significant disruption of BBB, as manifested by decreased TEER (by ~26%) and a parallel increased paracellular permeability to FITC- (4kDa) dextrans through hCMEC/D3 monolayers. The inhibition of Omi/HtrA2 by UCF-101 or Omi/HtrA2 shRNA reduced LPS-induced brain endothelial cell apoptosis, and resulted in significant improvement on LPS-induced BBB disruption as well as decreased occludin, claudin-5 and ZO-1 expressions. Omi/HtrA2 manipulated endothelial cell apoptosis by shifting into cytosol and inducing X-linked inhibitor of apoptosis protein (XIAP) degradation. UCF-101 administration or Omi/HtrA2 shRNA intervention did attenuate the degradation of XIAP, Poly ADP-ribose polymerase (PARP) cleavage, and caspase-3 cleavage. However, only UCF-101 partly prevented the mobilization of Omi/HtrA2 from the mitochondria to the cytosol after LPS intervention. That abrogation of Omi/HtrA2 by UCF-101 or Omi/HtrA2 shRNA resulted in a significant improvement on LPS-induced decrease of MMP. Oxidative stress was significantly increased in the LPS treated group compared to the control or NC-shRNA group. However, abrogation of Omi/HtrA2 by UCF-101 or Omi/HtrA2 shRNA did not significantly improve oxidative injury.Conclusions: Our study indicated an important role of Omi/HtrA2 in manipulating LPS-induced cell apoptosis and BBB integrity by translocating from mitochondria into cytosol in brain endothelial cells. Omi/HtrA2 induced mitochondrial pathway apoptosis, which involves inhibition of an important antiapoptotic protein XIAP and influence on MMP. Therapeutic methods that inhibit Omi/HtrA2 function may provide a novel therapeutic measure to septic encephalopathy.

Induction of C‐C motif chemokine ligand 2 through Toll‐like receptor 3 signaling in human cerebral microvascular endothelial cell/D3 cells: possible regulation by nuclear factor‐κB

AbstractObjectiveC‐C motif chemokine ligand 2 (CCL2) is a potent chemokine that plays an important role in host defense and inflammatory diseases in the central nervous system. Toll‐like receptor 3 (TLR3) is a pattern recognition receptor that recognizes viral double‐stranded RNAs. In the present study, we examined whether a TLR3 agonist induces CCL2 expression in brain microvascular endothelial cells, and investigated the mechanism underlying upregulation of CCL2 expression through TLR3 signaling.MethodsA human brain microvascular endothelial cell line, human cerebral microvascular endothelial cell (hCMEC)/D3 was treated with a TLR3 agonist, polyinosinic‐polycytidylic acid (poly IC). The mRNA and protein expression of CCL2 were evaluated using real‐time reverse transcription polymerase chain reaction and enzyme‐linked immunosorbent assay, respectively. The role of TLR3 and nuclear factor‐κB (NF‐κB) were examined with RNA interference. We also examined the effect of pretreatment with an NF‐κB inhibitor, SN50, an interferon regulatory factor 3 inhibitor MR67307 and human type I interferon neutralizing antibody mixture on poly IC‐induced CCL2 expression.ResultsExpression of CCL2 was induced by poly IC. Poly IC‐induced CCL2 expression was decreased by knockdown of TLR3 or NF‐κB p65 and by pretreatment with SN50. Neither MR67307 nor human type I interferon neutralizing antibody mixture affected the CCL2 expression induced by poly IC.ConclusionsCCL2 expression was induced by poly IC through TLR3 signaling, and NF‐κB is involved in this reaction. CCL2 produced by human brain endothelial cells might induce the infiltration of monocytes followed by immune and inflammatory reactions in the brain, and could be involved in the pathogenesis of viral encephalitis.

Human Brain Endothelial CXCR2 is Inflammation-Inducible and Mediates CXCL5- and CXCL8-Triggered Paraendothelial Barrier Breakdown.

Chemokines (C-X-C) motif ligand (CXCL) 5 and 8 are overexpressed in patients with multiple sclerosis, where CXCL5 serum levels were shown to correlate with blood–brain barrier dysfunction as evidenced by gadolinium-enhanced magnetic resonance imaging. Here, we studied the potential role of CXCL5/CXCL8 receptor 2 (CXCR2) as a regulator of paraendothelial brain barrier function, using the well-characterized human cerebral microvascular endothelial cell line hCMEC/D3. Low basal CXCR2 mRNA and protein expression levels in hCMEC/D3 were found to strongly increase under inflammatory conditions. Correspondingly, immunohistochemistry of brain biopsies from two patients with active multiple sclerosis revealed upregulation of endothelial CXCR2 compared to healthy control tissue. Recombinant CXCL5 or CXCL8 rapidly and transiently activated Akt/protein kinase B in hCMEC/D3. This was followed by a redistribution of tight junction-associated protein zonula occludens-1 (ZO-1) and by the formation of actin stress fibers. Functionally, these morphological changes corresponded to a decrease of paracellular barrier function, as measured by a real-time electrical impedance-sensing system. Importantly, preincubation with the selective CXCR2 antagonist SB332235 partially prevented chemokine-induced disturbance of both tight junction morphology and function. We conclude that human brain endothelial CXCR2 may contribute to blood–brain barrier disturbance under inflammatory conditions with increased CXCL5 and CXCL8 expression, where CXCR2 may also represent a novel pharmacological target for blood–brain barrier stabilization.

Transcriptional profiles of cytokines and chemokines reveal important pro-inflammatory response from endothelial cells during Orientia tsutsugamushi infection

Endothelial cells (EC) are key targets during Orientia tsutsugamushi infection. Knowledge of the pro-inflammatory response against O. tsutsugamushi by ECs is limited. The aim of the present study was to characterize the pro-inflammatory transcriptional response during the first 24 h of infection of the human dermal microvascular endothelial cell line with O. tsutsugamushi Karp by examining five-time points. The transcriptional profiles of 84 genes including cytokines, chemokines, growth factors, and TNF receptor superfamily genes were studied using a RT-PCR array. We identified 40 of the 84 genes that were up or down modulated during the early O. tsutsugamushi infection that differed remarkably from genes of non-infected cells. The modulated genes included: the interleukins (IL-1α/β, IL-4, IL-6, IL-7, IL-10, IL-11, IL-18, and IL-24), chemokines (CXCL8, CCL2/MCP1, CCL5/RANTES, and CCL17), growth factors (NODAL, CNTF, and CSF2/GM-CSF), and TNFSF13B. IL-1β, IL-4, and IL-11 were highly induced at one hour post infection, whereas, CCL17 was profoundly up-regulated and IFNα2 was greatly down-regulated during the entire 24-hour time course. These results provide insight into the early pro-inflammatory response of endothelial cells to O. tsutsugamushi infection and indicate their potential role in the pathophysiology of the host's initial response to O. tsutsugamushi infection.

Simultaneous determination of intracellular concentrations of tenofovir, emtricitabine, and dolutegravir in human brain microvascular endothelial cells using liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Combination antiretroviral therapy (cART) regimens are recommended for HIV patients to better achieve and maintain plasma viral suppression. Despite adequate plasma viral suppression, HIV persists inside the brain, which is, in part thought to result from poor brain penetration of antiretroviral drugs. In this study, a simple and ultra-sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous determination of tenofovir, emtricitabine, and dolutegravir in cell lysates of an immortalized human brain microvascular endothelial cell line (hCMEC/D3) was developed and validated. Analytes were separated on a reverse phase C18 column using water and 0.1% formic acid in acetonitrile as mobile phases. The analytes were detected using positive electrospray ionization mode with multiple reaction monitoring (MRM). The assay was linear in the concentration range of 0.1–100 ng mL−1 for all analytes. Intra- and inter-assay precision and accuracy were within ±13.33% and ±10.53%, respectively. This approach described herein was used to determine the intracellular accumulation of tenofovir, emtricitabine, dolutegravir simultaneously in hCMEC/D3 cells samples.

Organic chemicals from diesel exhaust particles affects intracellular calcium, inflammation and β-adrenoceptors in endothelial cells

Exposure to diesel exhaust particles (DEP) may contribute to endothelial dysfunction and cardiovascular disease. DEP, extractable organic material from DEP (DEP-EOM) and certain PAHs seem to trigger [Ca2+]i increase as well as inflammation via GPCRs like βARs and PAR-2. In the present study we explored the involvement of βARs and PAR-2 in effects of DEP-EOM on [Ca2+]i and expression of inflammation-associated genes in the endothelial cell-line HMEC-1. We exposed the human microvascular endothelial cell line HMEC-1 to DEP-EOM fractionated by sequential extraction with solvents of increasing polarity: n-hexane (n-Hex-EOM), dichloromethane (DCM-EOM), methanol (Methanol-EOM) and water (Water-EOM). While Methanol-EOM and Water-EOM had no marked effects, n-Hex-EOM and DCM-EOM enhanced [Ca2+]i (2–3 times baseline) and expression of inflammation-associated genes (IL-1α, IL-1β, COX-2 and CXCL8; 2–15 times baseline) in HMEC-1. The expression of βARs (60–80% of baseline) and βAR-inhibitor carazolol suppressed the increase in [Ca2+]i induced by both n-Hex- and DCM-EOM. Carazolol as well as the Ca2+-channel inhibitor SKF-96365 reduced the DCM-EOM-induced pro-inflammatory gene-expression. Overexpression of βARs increased DCM-EOM-induced [Ca2+]i responses in HEK293 cells, while βAR-overexpression suppressed [Ca2+]i responses from n-Hex-EOM. Furthermore, the PAR-2-inhibitor ENMD-1068 attenuated [Ca2+]i responses to DCM-EOM, but not n-Hex-EOM in HMEC-1.The results suggest that βAR and PAR-2 are partially involved in effects of complex mixtures of chemicals extracted from DEP on calcium signalling and inflammation-associated genes in the HMEC-1 endothelial cell-line.

Cellular recycling-driven in vivo half-life extension using recombinant albumin fusions tuned for neonatal Fc receptor (FcRn) engagement

Recombinant albumin-drug genetic fusions are an effective technology to prolong the serum half-life of therapeutics that has resulted in marketed products. Indirect evidence suggests albumin fusions' long circulation is controlled by engagement with the cellular recycling neonatal Fc receptor (FcRn) in addition to reduced kidney filtration. In this work, we have used a panel of recombinant fusions, engineered with different human FcRn (hFcRn) affinity, including a novel high binding albumin variant (HBII), to directly define and importantly, control the intracellular mechanism as a half-life extension tuning method. mNeonGreen or mCherry fusion to the N-terminal of the recombinant human albumin (rHA) variants null-binder (rHA NB), wild-type (rHA WT), high-binder I (rHA HBI), and high-binder II (rHA HBII) did not generally interfere with hFcRn interaction determined by Biolayer Interferometry. Co-localisation of the albumins with endosomal, but not lysosomal, markers was shown by confocal microscopy for high, but not low, hFcRn binders in a human microvascular endothelial hFcRn overexpressing cell line (HMEC-1 FcRn) suggestive of endosomal compartmentalisation. Furthermore, a cellular recycling assay revealed increased recycling of albumin fusions for the high binding variants (mNeonGreen WT; ~1, mNeonGreen HBI; 5.26-fold higher, and mNeonGreen HBII; 5.77-fold higher) in the hFcRn overexpressing cell line. In vivo experiments demonstrated a direct in vitro recycling/in vivo half-life correlation with a longer circulation for the mCherry fusions engineered with high hFcRn affinity that was highest with the HBII variant of 30.1 h compared to 18.2 h for the mCherry WT. This work gives the first direct evidence for an FcRn-driven endosomal cellular recycling pathway for recombinant albumin fusions that correlates with half-life extension controlled by the affinity to hFcRn; promoting a versatile method to tune the pharmacokinetics of albumin fusion-based therapeutics not met by current technologies.

Macrophage Migration Inhibitory Factor-Induced Autophagy Contributes to Thrombin-Triggered Endothelial Hyperpermeability in Sepsis.

Vascular leakage contributes to the high morbidity and mortality associated with sepsis. Exposure of the endothelium to inflammatory mediators, such as thrombin and cytokines, during sepsis leads to hyperpermeability. We recently observed that autophagy, a cellular process for protein turnover, is involved in macrophage migration inhibitory factor (MIF)-induced endothelial hyperpermeability. Even though it is known that thrombin induces endothelial cells to secrete MIF and to increase vascular permeability, the possible role of autophagy in this process is unknown. In this study, we proposed and tested the hypothesis that MIF-induced autophagy plays an important role in thrombin-induced endothelial hyperpermeability. We evaluated the effects of thrombin on endothelial permeability, autophagy induction, and MIF secretion in vitro using the human microvascular endothelial cell line-1 and human umbilical vein endothelial cells. Several mechanisms/read outs of endothelial permeability and autophagy formation were examined. We observed that blocking autophagy attenuated thrombin-induced endothelial hyperpermeability. Furthermore, thrombin-induced MIF secretion was involved in this process because MIF inhibition reduced thrombin-induced autophagy and hyperpermeability. Finally, we showed that blocking MIF or autophagy effectively alleviated vascular leakage and mortality in endotoxemic mice. Thus, MIF-induced autophagy may represent a common mechanism causing vascular leakage in sepsis.

Fingolimod promotes blood-nerve barrier properties in vitro.

ObjectiveThe main effect of fingolimod is thought to be functional antagonism of lymphocytic S1P1 receptors and the prevention of lymphocyte egress from lymphoid tissues, thereby reducing lymphocyte infiltration into the nervous system. However, a growing number of reports suggest that fingolimod also has a direct effect on several cell types in the nervous system. Although we previously reported that fingolimod enhances blood–brain barrier (BBB) functions, there have been no investigations regarding the blood–nerve barrier (BNB). In this study, we examine how fingolimod affects the BNB.MethodsAn immortalized human peripheral nerve microvascular endothelial cell line (HPnMEC) was used to evaluate BNB barrier properties. We examined tight junction proteins and barrier functions of HPnMECs in conditioned medium with or without fingolimod‐phosphate and blood sera from patients with typical chronic inflammatory demyelinating polyneuropathy (CIDP).ResultsIncubation with fingolimod‐phosphate increased levels of claudin‐5 mRNA and protein as well as TEER values in HPnMECs. Conversely, typical CIDP sera decreased claudin‐5 mRNA/protein levels and TEER values in HPnMECs; however, pretreatment with fingolimod‐phosphate inhibited the effects of the typical CIDP sera.ConclusionsFingolimod‐phosphate directly modifies the BNB and enhances barrier properties. This mechanism may be a viable therapeutic target for CIDP, and fingolimod may be useful in patients with typical CIDP who have severe barrier disruption.

Ionophore and Biometal Modulation of P-glycoprotein Expression and Function in Human Brain Microvascular Endothelial Cells.

Biometals such as zinc and copper have been shown to affect tight junction expression and subsequently blood-brain barrier (BBB) integrity. Whether these biometals also influence the expression and function of BBB transporters such as P-glycoprotein (P-gp) however is currently unknown. Using the immortalised human cerebral microvascular endothelial (hCMEC/D3) cell line, an in-cell western assay (alongside western blotting) assessed relative P-gp expression after treatment with the metal ionophore clioquinol and biometals zinc and copper. The fluorescent P-gp substrate rhodamine-123 was employed to observe functional modulation, and inductively coupled plasma mass spectrometry (ICP-MS) provided information on biometal trafficking. A 24-h treatment with clioquinol, zinc and copper (0.5, 0.5 and 0.1μM) induced a significant upregulation of P-gp (1.7-fold) assessed by in-cell western and this was confirmed with western blotting (1.8-fold increase). This same treatment resulted in a 23% decrease in rhodamine-123 accumulation over a 1h incubation. ICP-MS demonstrated that while t8his combination treatment had no effect on intracellular zinc concentrations, the treatment significantly enhanced bioavailable copper (4.6-fold). Enhanced delivery of copper to human brain microvascular endothelial cells is associated with enhanced expression and function of the important efflux pump P-gp, which may provide therapeutic opportunities for P-gp modulation.

Novel Brassinosteroid-Modified Polyethylene Glycol Micelles for Controlled Release of Agrochemicals.

Two synthetic analogues of brassinosteroids (DI31 and S7) exhibit good plant growth enhancer activity. However, their hydrophobicity and quick metabolism in plants have limited their application and benefits in agriculture. Our objective was to prepare novel brassinosteroid-modified polyethylene glycol (PEG) micelles to achieve controlled release with extended stability while retaining agrochemical activity. Spectroscopic studies confirmed quantitative disubstitution of studied PEGs with the brassinosteroids, while elemental analysis assessed purity of the synthesized conjugates. Conjugates were also characterized by X-ray diffraction and thermal analysis. Dynamic and static light scattering showed stable and homogeneous approximately spherical micelles with average hydrodynamic diameters of 22-120 nm and almost neutral ζ potential. Spherical 30-140 nm micelles were observed by electron microscopy. Sustained in vitro releases at pH 5.5 were extended up to 96 h. Prepared PEG micelles showed good agrochemical activity in the radish seed bioassay and no cytotoxicity to the human microvascular endothelial cell line in the MTS test.

Extracellular Vesicles Secreted by Human Urine-Derived Stem Cells Promote Ischemia Repair in a Mouse Model of Hind-Limb Ischemia

Background/Aims: Our previous studies have shown that human urine-derived stem cells (USCs) have great potential as a cell source for cytotherapy and tissue engineering and that extracellular vesicles (EVs) secreted by USCs (USCs-EVs) can prevent diabetes-induced kidney injury in an animal model. The present study was designed to evaluate the effects of USCs-EVs on ischemia repair. Methods: USCs-EVs were isolated and purified by a battery of centrifugation and filtration steps. The USCs-EVs were then characterized by transmission electron microscopy, western blot and tunable resistive pulse sensing techniques. After intramuscularly transplanting USCs-EVs into an ischemic mouse hind-limb, we observed the therapeutic effects of USCs-EVs on perfusion by laser doppler perfusion imaging, angiogenesis and muscle regeneration by histology and immunohistochemistry techniques over 21 days. We subsequently tested whether USCs-EVs can induce the proliferation of a human microvascular endothelial cell line HMEC-1 and a mouse myoblast cell line C2C12 by cell counting kit 8 assay in vitro. Meanwhile, the potential growth factors in the USCs-EVs and supernatants of the USCs cultures were detected by enzyme-linked immunosorbent assay. Results: The USCs-EVs were spherical vesicles with a diameter of 30–150 nm and expressed exosomal markers, such as CD9, CD63 and Tsg101. Ischemic limb perfusion and function were markedly increased in the hind-limb ischemia (HLI) model after USCs-EVs administration. Moreover, angiogenesis and muscle regeneration levels were significantly higher in the USCs-EVs treatment group than in the PBS group. The in vitro experiments showed that USCs-EVs facilitated HMEC-1 and C2C12 cell proliferation in a dose-dependent manner. Conclusions: These results revealed for the first time that USCs-EVs efficiently attenuate severe hind-limb ischemic injury and represent a novel therapy for HLI.

High dengue virus load differentially modulates human microvascular endothelial barrier function during early infection.

Plasma leakage is the main pathophysiological feature in severe dengue, resulting from altered vascular barrier function associated with an inappropriate immune response triggered upon infection. The present study investigated functional changes using an electric cell-substrate impedance sensing system in four (brain, dermal, pulmonary and retinal) human microvascular endothelial cell (MEC) lines infected with purified dengue virus, followed by assessment of cytokine profiles and the expression of inter-endothelial junctional proteins. Modelling of changes in electrical impedance suggests that vascular leakage in dengue-infected MECs is mostly due to the modulation of cell-to-cell interactions, while this loss of vascular barrier function observed in the infected MECs varied between cell lines and DENV serotypes. High levels of inflammatory cytokines (IL-6 and TNF-α), chemokines (CXCL1, CXCL5, CXCL11, CX3CL1, CCL2 and CCL20) and adhesion molecules (VCAM-1) were differentially produced in the four infected MECs. Further, the tight junctional protein, ZO-1, was down-regulated in both the DENV-1-infected brain and pulmonary MECs, while claudin-1, PECAM-1 and VE-cadherin were differentially expressed in these two MECs after infection. Non-purified virus stock was also studied to investigate the impact of virus stock purity on dengue-specific immune responses, and the results suggest that virus stock propagated through cell culture may include factors that mask or alter the DENV-specific immune responses of the MECs. The findings of the present study show that high DENV load differentially modulates human microvascular endothelial barrier function and disrupts the function of inter-endothelial junctional proteins during early infection with organ-specific cytokine production.

Effects of cognate, non-cognate and synthetic CXCR4 and ACKR3 ligands on human lung endothelial cell barrier function

Recent evidence suggests that chemokine CXCL12, the cognate agonist of chemokine receptors CXCR4 and ACKR3, reduces thrombin-mediated impairment of endothelial barrier function. A detailed characterization of the effects of CXCL12 on thrombin-mediated human lung endothelial hyperpermeability is lacking and structure-function correlations are not available. Furthermore, effects of other CXCR4/ACKR3 ligands on lung endothelial barrier function are unknown. Thus, we tested the effects of a panel of CXCR4/ACKR3 ligands (CXCL12, CXCL11, ubiquitin, AMD3100, TC14012) and compared the CXCR4/ACKR3 activities of CXCL12 variants (CXCL12α/β, CXCL12(3–68), CXCL121, CXCL122, CXCL12-S-S4V, CXCL12-R47E, CXCL12-K27A/R41A/R47A) with their effects on human lung endothelial barrier function in permeability assays. CXCL12α enhanced human primary pulmonary artery endothelial cell (hPPAEC) barrier function, whereas CXCL11, ubiquitin, AMD3100 and TC14012 were ineffective. Pre-treatment of hPPAEC with CXCL12α and ubiquitin reduced thrombin-mediated hyperpermeability. CXCL12α-treatment of hPPAEC after thrombin exposure reduced barrier function impairment by 70% (EC50 0.05–0.5nM), which could be antagonized with AMD3100; ubiquitin (0.03–3μM) was ineffective. In a human lung microvascular endothelial cell line (HULEC5a), CXCL12α and ubiquitin post-treatment attenuated thrombin-induced hyperpermeability to a similar degree. CXCL12(3–68) was inefficient to activate CXCR4 in Presto-Tango β-arrestin2 recruitment assays; CXCL12-S-S4V, CXCL12-R47E and CXCL12-K27A/R41A/R47A showed significantly reduced potencies to activate CXCR4. While the potencies of all proteins in ACKR3 Presto-Tango assays were comparable, the efficacy of CXCL12(3–68) to activate ACKR3 was significantly reduced. The potencies to attenuate thrombin-mediated hPPAEC barrier function impairment were: CXCL12α/β, CXCL121, CXCL12-K27A/R41A/R47A > CXCL12-S-S4V, CXCL12-R47E > CXCL122 > CXCL12(3–68). Our findings indicate that CXCR4 activation attenuates thrombin-induced lung endothelial barrier function impairment and suggest that protective effects of CXCL12 are dictated by its CXCR4 agonist activity and interactions of distinct protein moieties with heparan sulfate on the endothelial surface. These data may facilitate development of compounds with improved pharmacological properties to attenuate thrombin-induced vascular leakage in the pulmonary circulation.

PSN-PC: A Novel Antimicrobial and Anti-Biofilm Peptide from the Skin Secretion of Phyllomedusa-camba with Cytotoxicity on Human Lung Cancer Cell.

Peptides derived from amphibian skin secretion are promising drug prototypes for combating widespread infection. In this study, a novel peptide belonging to the phylloseptin family of antimicrobial peptides was isolated from the skin secretion of the Phyllomedusa camba, namely phylloseptin-PC (PSN-PC). The biosynthetic precursor was obtained by molecular cloning and the mature peptide sequence was confirmed through tandem mass spectrometry (MS/MS) fragmentation sequencing in the skin secretion. The synthetic replicate exhibited a broad spectrum antimicrobial activity against Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans at concentrations of 2, 2, 8, 32 and 2 µM, respectively. It also showed the capability of eliminating S. aureus biofilm with a minimal biofilm eradication concentration of 8 µM. The haemolysis of this peptide was not significant at low concentrations but had a considerable increase at high concentrations. Additionally, this peptide showed an anti-proliferation effect on the non-small cell lung cancer cell line (NCI-H157), with low cytotoxicity on the human microvascular endothelial cell line (HMEC-1). The discovery of the novel peptide may provide useful clues for new drug discoveries.

Long-term exposure to ethanol downregulates tight junction proteins through the protein kinase Cα signaling pathway in human cerebral microvascular endothelial cells.

Brain microvascular endothelial cells (BMECs) are the primary component of the blood-brain barrier (BBB). Tight junction (TJ) proteins, including claudin, occludin and zonula occludens (ZO)-1, ZO-2 and ZO-3, maintain the structural integrity of BMECs. Ethanol activates the assembly and disassembly of TJs, which is a process that is regulated by protein kinase C (PKC). In addition, ethanol treatment leads to the loss of structural integrity, which damages the permeability of the BBB and subsequently affects central nervous system homeostasis, thus allowing additional substances to enter the brain. However, the mechanisms underlying ethanol-induced loss of BBB structure remain unknown. It has been hypothesized that long-term exposure to ethanol reduces the expression of claudin-5, occludin and ZO-1 via the PKC signaling pathway, thereby affecting BBB structural integrity. In the current study, the human cerebral microvascular endothelial cell line, HCMEC/D3, was treated with 50, 100, 200 and 400 mM ethanol for 24, 48 and 72 h. Cell viability was determined using an MTS assay. The expression of claudin-5, occludin and ZO-1 protein and mRNA was measured using western blot analysis and reverse transcription-quantitative polymerase chain reaction, respectively. Following the pretreatment of HCMEC/D3 cells with the PKCα-specific inhibitor, safingol (10 µmol/l), the expression of claudin-5, occludin, ZO-1 and phosphorylated (p)-PKCα was measured using western blot analysis, and PKCα localization was determined by immunofluorescence. With increasing concentrations of ethanol, the expression of claudin-5, occludin and ZO-1 protein decreased, while the expression of claudin-5, occludin and ZO-1 mRNA increased. Exposure to ethanol significantly increased the expression of p-PKCα, whereas no significant effect on the expression of PKCα was observed. Following 48 h treatment with 200 mM ethanol, the expression of claudin-5, occludin and ZO-1 protein was significantly decreased when compared with the control. By contrast, the expression of p-PKCα was increased, and increased translocation of PKCα from the cytoplasm to the nuclear membrane and nucleus was observed. In addition, the results demonstrated that safingol significantly reversed these effects of ethanol. In conclusion, long-term exposure to ethanol downregulates the expression of claudin-5, occludin and ZO-1 protein in HCMEC/D3 s, and this effect may be mediated via activation of PKCα.