Cultures Of Umbilical Vein Endothelial Cells Research Articles

Effect of ascorbic acid, α-tocopherol and its combination on human umbilical vein endothelial cells permeability exposed to high concentration of glucose

Aim — To evaluate the effect of ascorbic acid, α-tocopherol, and its combination on the permeability of human umbilical vein endothelial cells (HUVEC’S) culture after high glucose concentration exposure. Material and Methods — The study was performed using HUVEC’S culture on transwell polyester filter. The ascorbic acid, α-tocopherol, and its combination (25-200 µM) were concomitantly given with glucose 22 µM to the culture media. After seven days of treatment, endothelial permeability was observed by measuring the concentration of trypan blue-labeled albumin in a luminal compartment with a spectrophotometer (λ=592 nm). The morphology of the cells was observed under an inverted microscope. The data was analyzed using ANOVA test followed by Dunnet test (p<0.05). Results — HUVEC’S culture exposed to high concentration glucose increased endothelial permeability better than those which exposed to normal concentration (p<0.05). The treatment of ascorbic acid 200 µM, α-tocopherol 100 µM and 200 µM were capable to inhibit the increase HUVEC’S permeability exposed to high concentration glucose better than control group (p<0.05). Moreover, two combinations of ascorbic acid and α-tocopherol, respectively 50 and 100 µM, could prevent the increased permeability of HUVEC’S culture better than control (p<0.05). Conclusion — Ascorbic acid, α-tocopherol, and its combination could inhibit the increase of endothelial permeability exposed to a high concentration of glucose.

Monitoring matrix remodeling in the cellular microenvironment using microrheology for complex cellular systems

Multiple particle tracking (MPT) microrheology was employed for monitoring the development of extracellular matrix (ECM) mechanical properties in the direct microenvironment of living cells. A customized setup enabled us to overcome current limitations: (i) Continuous measurements were enabled using a cell culture chamber, with this, matrix remodeling by fibroblasts in the heterogeneous environment of macroporous scaffolds was monitored continuously. (ii) Employing tracer laden porous scaffolds for seeding human mesenchymal stem cells (hMSCs), we followed conventional differentiation protocols. Thus, we were, for the first time able to study the massive alterations in ECM elasticity during hMSC differentiation. (iii) MPT measurements in 2D cell cultures were enabled using a long distance objective. Exemplarily, local mechanical properties of the ECM in human umbilical vein endothelial cell (HUVEC) cultures, that naturally form 2D layers, were investigated scaffold-free.Using our advanced setup, we measured local, apparent elastic moduli G0,app in a range between 0.08 and 60 Pa. For fibroblasts grown in collagen-based scaffolds, a continuous decrease of local matrix elasticity resulted during the first 10 hours after seeding. The osteogenic differentiation of hMSC cells cultivated in similar scaffolds, led to an increase of G0,app by 100 %, whereas after adipogenic differentiation it was reduced by 80 %. The local elasticity of ECM that was newly secreted by HUVECs increased significantly upon addition of protease inhibitor and in high glucose conditions even a twofold increase in G0,app was observed.The combination of these advanced methods opens up new avenues for a broad range of investigations regarding cell-matrix interactions and the propagation of ECM mechanical properties in complex biological systems. Statement of SignificanceCells sense the elasticity of their environment on a micrometer length scale. For studying the local elasticity of extracellular matrix (ECM) in the direct environment of living cells, we employed an advanced multipleparticle tracking microrheology setup. MPT is based on monitoring the Brownian motion oftracer particles, which is restricted by the surrounding network. Network elasticity can thusbe quantified.Overcoming current limitations, we realized continuous investigations of ECM elasticityduring fibroblast growth. Furthermore, MPT measurements of stem cell ECM showed ECMstiffening during osteogenic differentiation and softening during adipogenic differentiation.Finally, we characterized small amounts of delicate ECM newly secreted in scaffold-freecultures of endothelial cells, that naturally form 2D layers.

Phenotypic and Functional Characterization of human Umbilical Vein Endothelial Cells (hUVEC) Cultures Established under Physiologic vs. Atmospheric Oxygen Tension

Primary cultures of human umbilical vein endothelial cells (hUVEC) have historically been established at atmospheric oxygen (O2) tension (21%) and their surface marker profiles and angiogenic potential in are understood. However, physiologic O2 tension ranges from ~1% in cartilage to ~13% in major arteries, thus 21% O2 represents a supraphysiologic O2 exposure. We conducted experiments to establish and characterize hUVEC at 21% O2 and physiologically relevant oxygen tension (5% O2) from three independent donors.hUVEC were isolated by standard methods with dissociation and culture steps conducted at 21% or 5% O2. Cells were maintained in standard endothelial cell growth media and were cryopreserved at the end of passage 1. Cells were thawed, used to establish live passage 2 cultures, and confirmed to be sterile and free of mycoplasma and endotoxin. hUVEC established at 5% O2 (hUVEC‐LO) were compared to hUVEC established at 21% O2 (hUVEC‐HO) with respect to population doubling time, surface marker expression by flow cytometry, and angiogenic potential by tube formation assay.Both hUVEC‐LO and hUVEC‐HO were immunopositive for CD31, VE‐cadherin, CD146, CD202b, and vEGFR1, and were negative for CD45 and CD90. hUVEC‐LO uniquely expressed a greater percentage of markers associated with an endothelial progenitor phenotype, including CD117, Slug, CD133, and vEGFR2. The calculated population doubling time of hUVEC‐LO was on average 10% faster than the donor‐matched hUVEC‐HO. Angiogenic potential was confirmed by tube formation assay for each lot at the oxygen tension at which they were established. Crossover experiments were conducted to assess tube formation in hUVEC‐LO and hUVEC‐HO under assay conditions of 21% or 5% O2. No statistical differences were noted after 6 hours of assay duration with respect to: number of junctions, segments, meshes, and total length of segments, branches, or extremities. Significant differences were observed beyond 6 hours between hUVEC‐LO and hUVEC‐HO in mean mesh size and number of branches, but the magnitude and pattern of differences varied from donor‐to‐donor. In contrast, the early phase of angiogenesis (0–6 hours) revealed notable differences between hUVEC‐LO and hUVEC‐HO, with hUVEC‐LO generally exhibiting greater variance with respect to number of junctions, mesh area, number of meshes, and number of segments.Taken together, these results suggest that hUVEC‐LO may have a more stem‐like phenotype characterized by the retention of progenitor‐associated surface markers and a faster doubling time compared to hUVEC‐HO from the same donor. While the ultimate capacity for tube formation did not differ between hUVEC‐LO and hUVEC‐HO assayed at 21% or 5% O2, the preliminary data suggest that hUVEC‐LO respond more actively to adapt to changes in oxygen tension, undergoing significant fluctuations in multiple measures at early time points. Further experiments are needed to elucidate the processes and mechanism(s) that lead to differential phenotype and/or performance of hUVEC‐LO and hUVEC‐HO, in particular during the early initiating events of angiogenesis.

HLA expression levels of unstimulated and cytokine stimulated human umbilical vein endothelial cells.

Transplant rejection occurs following recipient recognition of mismatched HLA on donor tissue, but active rejection is dependent not only upon the severity of the T cell or alloantibody response, but also upon the cell surface expression of target HLA molecules. To investigate the variation in HLA expression using a model of endothelium, human umbilical vein endothelial cell (HUVEC) cultures were generated from 48 umbilical cords donated consecutively following planned caesarean section. HUVECs were stimulated using the cytokines tumour necrosis factor alpha and interferon gamma and HLA expression of unstimulated and stimulated cells determined using flow cytometry. HLA-A2, HLA-A3 and HLA-C antigens all showed a modest increase in expression for 12 hours post cell activation, followed by a more pronounced response over the next 24 to 36 hours. Each of these antigens increased by up to 40 times over unstimulated levels and in addition cells homozygous for specific HLA antigens on average had twice the amount of antigen expressed compared with cells heterozygous for that antigen, both when unstimulated and following cytokine stimulation. Cell activation is an important consideration in the assessment of transplant risk and may help progress towards understanding why rejection does not always occur in the presence of significant donor specific antibody. This data also confirms guidelines for transplantation, which recommend doubling the specific antibody level when considering immunological risk for homozygous donors.

A dynamic culture platform enhances the efficiency of the 3D HUVEC-based tube formation assay.

Cell-based in vitro biological models traditionally use monolayer cell cultures grown over plastic surfaces bathing in static media. Higher fidelity to a natural biological tissue is expected to result from growing the cells in a three-dimensional (3D) matrix. However, due to the decreased rate of diffusion inherent to increased distances within a tridimensional space, proper fluidic conditions are needed in this setting to better approximate a physiological environment. To this aim, we here propose a prototypal dynamic cell culture platform for the automatic medium replacement, via periodic perfusion flow, in a human umbilical vein endothelial cell (HUVECs) culture seeded in a Geltrex™ matrix. A state-of-the-art angiogenesis assay performed in these dynamic conditions showed sizable effects with respect to conventional static control cultures, with significantly enhanced pro-(dual antiplatelet therapy [DAPT]) and anti-(EDTA) angiogenic compound activity. In particular, dynamic culture conditions (a) enhance the 3D-organization of HUVECs into microtubule structure; (b) accelerate and improve endothelial tube formation by HUVECs in the presence of DAPT; (c) are able to completely revert the blocking effects of EDTA. These evidence emphasize the need of setting proper fluidic conditions for a better approximation of a physiological environment as an appropriate evolution of current cell culture paradigms.

CYP1A1, GSTT1, IL-6 and IL-8 transcription and IL-6 secretion on umbilical endothelial cells from hypertensive pregnant women: Preliminary results.

The impact of pregnancy hypertension in the offspring endothelia remains unknown. We evaluated the transcriptional expression of four genes that participate in the process of endothelial dysfunction using umbilical vein endothelial cell cultures (HUVEC) from healthy pregnant women (PW) and those with hypertensive disorders (HD). The cytochrome P450 1A1 (CYP1A1), gluthathione S-transferase subtype T1 (GSTT1), interleukin 6 (IL-6) and 8 (IL-8) mRNA and IL-6 protein levels were assessed. IL-6 and IL-8 transcripts were significantly reduced in HUVEC obtained from HD women. Our results suggest that a hypertensive environment in utero modifies the transcriptional expression of key inflammatory molecules in the newborn.

Magnesium sulfate inhibits inflammation through P2X7 receptors in human umbilical vein endothelial cells.

Introduction:Magnesium sulfate (MgSO4) is utilized for fetal neuroprotection in preterm birth but its mechanism of action is still poorly understood. P2X7 receptor (P2X7R) is required for secretion of IL-1β, and can be blocked by divalent cations such as magnesium (Mg) and its own antagonist, Brilliant Blue G (BBG). We sought to determine, whether during inflammation MgSO4 can block endothelial IL-1β secretion, using an in-vitro model.Methods:Human umbilical vein endothelial cell (HUVEC) cultures were treated with varying doses of LPS, 2’(3)-O-(4-Benzoylbenzoyl) adenosine-5’-triphosphate (BzATP), BBG and MgSO4 for 3- or 24-hours. We determined cell cytotoxicity, apoptosis, IL-1β mRNA expression, IL-1β production and secretion and P2X7R expression on HUVECs.Results:We demonstrated that MgSO4 is efficacious in blocking IL-1β-mediated-inflammation in HUVECs, at both the initiation and propagation phases of inflammation. MgSO4 exerts these anti-inflammatory effects via downregulation of P2X7Rs on HUVECs.Conclusion:LPS-exposure increases IL-1β production and secretion in HUVECs, which is further intensified by P2X7R agonist, BzATP while MgSO4 inhibits IL-1β in both presence and absence of BzATP. This effect is similar to the results of P2X7R antagonist, BBG, suggesting that the anti-inflammatory effects of MgSO4is through P2X7R.

The angiotensin-(1-7)/Mas receptor axis protects from endothelial cell senescence via klotho and Nrf2 activation.

Endothelial cell senescence is a hallmark of vascular aging that predisposes to vascular disease. We aimed to explore the capacity of the renin–angiotensin system (RAS) heptapeptide angiotensin (Ang)‐(1‐7) to counteract human endothelial cell senescence and to identify intracellular pathways mediating its potential protective action. In human umbilical vein endothelial cell (HUVEC) cultures, Ang II promoted cell senescence, as revealed by the enhancement in senescence‐associated galactosidase (SA‐β‐gal+) positive staining, total and telomeric DNA damage, adhesion molecule expression, and human mononuclear adhesion to HUVEC monolayers. By activating the G protein‐coupled receptor Mas, Ang‐(1‐7) inhibited the pro‐senescence action of Ang II, but also of a non‐RAS stressor such as the cytokine IL‐1β. Moreover, Ang‐(1‐7) enhanced endothelial klotho levels, while klotho silencing resulted in the loss of the anti‐senescence action of the heptapeptide. Indeed, both Ang‐(1‐7) and recombinant klotho activated the cytoprotective Nrf2/heme oxygenase‐1 (HO‐1) pathway. The HO‐1 inhibitor tin protoporphyrin IX prevented the anti‐senescence action evoked by Ang‐(1‐7) or recombinant klotho. Overall, the present study identifies Ang‐(1‐7) as an anti‐senescence peptide displaying its protective action beyond the RAS by consecutively activating klotho and Nrf2/HO‐1. Ang‐(1‐7) mimetic drugs may thus prove useful to prevent endothelial cell senescence and its related vascular complications.

Ambon Banana-Tree Sap stimulated Endothelial Cell Migration in Human Umbilical Vein Endothelial Cells (HUVECs) Induced with Inflammatory Mediator IL-1

Ambon banana-tree ( Musa paradisiaca var.sapientum) sap (ABT sap) contains active substances that can stimulate the wound healing process such as: flavonoid, saponin, ascorbic acid. Those substances are needed in an important phase of wound healing called angiogenesis through activation of Vascular Endothelial Growth Factor (VEGF) pathway that stimulates actin reorganization in endothelial cell migration and proliferation. The aim of this study is to evaluate the effectiveness of banana-tree sap in stimulating cells migration, VEGF and nitric oxide (NO) level in Human Umbilical Vein Endothelial Cells (HUVECs) culture that induced by pro-inflammatory cytokine IL-1 (Interleukin-1). The ABT sap was obtained by pressing a fresh Ambon banana stem, then was mixed with culture medium into 0.125%, 0.25% and 0.5% concentration. The ABT sap then was used for treating HUVECs cell that induced by IL-1 200 ng/mL for 48-hours. The result of this study shows that Ambon banana-tree sap increased HUVECs cell migration. However, after 48-hours treatment with ABT sap showed no difference in the VEGF and NO level in the culture media. These results suggested ABT sap able to increase migration to HUVECs that promising for wound healing agent. Therefore, the further research to elucidate the detailed mechanism and identify the active compound warrant to be done.

Increased SERUM VON Willebrand Levels in Chronic Lymphocytic Leukemia (CLL) Patients Are Related to a Shorter Time to Treatment

CLL is a frequent and usually indolent disease that require only follow-up in about 2/3 of patients while the rest 1/3 of patients are symptomatic, have a worse outcome and need to be treated. The role of angiogenesis in CLL prognosis remains unclear. It has been shown that CLL plasma promotes von Willebrand factor (vWF) secretion, and expression in human umbilical vein endothelial cell cultures (HUVECs) [1]. Therefore, we investigated whether serum vWF levels in CLL patients at diagnosis could provide prognostic information.Patients and methods: 33 CLL patients were studied of whom 55%, 32% and 13% were Binet staged A, B and C respectively. 45% never required treatment while 55% required treatment either at the time of diagnosis or during their follow-up. The time to first treatment (TFT) was 34 months. Serum vWF was measured by ELISA (R&D Quantiquine, duo Set) in frozen sera drawn at patients' diagnosis and in 10 healthy individuals (HI). Measurements were performed in duplicate according to the manufacturer's instructions. Statistical analysis was performed by conventional methods, using the SPSS v.22 software. P values below 0,05 were considered statistically significant.Results: Serum vWF levels ranged from 178,5 to 14353,5 pg/ml (median 1028,5) in patients and from 374,6 to 1141,3 pg/ml (median 528,5) in HI, the difference being statistically significant (p< 0,05). Patients with Serum vWF levels above 528,5 pg/ml (median normal value) had a significantly shorter TFT than the others (p=0,01) as shown in figure. Otherwise, serum vWF levels correlated only with hypogammaglobulinemia (p=0,04).Conclusion: Serum vWF levels were increased in CLL patients. Importantly, we showed for the first time, to our knowledge, that higher vWF levels correlated with a shorter TFT, suggesting that increased vWF levels reflect active neoangiogenesis that in turn, contributes to a more aggressive disease behavior.[1] Shahidi M et al, Induction of endothelial cell proliferation and von Willebrand factor expression and secretion by leukemic plasma of patients with chronic lymphocytic leukemia before and after inhibition of NF-κB. Blood Coagul Fibrinolysis. 2016 Sep;27(6):711-6. DisclosuresNo relevant conflicts of interest to declare.

An effective "three-in-one" screening assay for testing drug and nanoparticle toxicity in human endothelial cells.

Evaluating nanoparticle (NP) toxicity in human cell systems is a fundamental requirement for future NP biomedical applications. In this study, we have designed a screening assay for assessing different types of cell death induced by NPs in human umbilical vein endothelial cell (HUVEC) culture. This assay consists of WST-8, LDH and Hoechst 33342 staining, all performed in one well, which enables an evaluation of cell viability, necrosis and apoptosis, respectively, in the same cell sample. The 96-well format and automated processing of fluorescent images enhances the assay rapidity and reproducibility. After testing the assay functionality with agents that induced different types of cell death, we investigated the endothelial toxicity of superparamagnetic iron oxide nanoparticles (SPIONs, 8 nm), silica nanoparticles (SiNPs, 7–14 nm) and carboxylated multiwall carbon nanotubes (CNTCOOHs, 60 nm). Our results indicated that all the tested NP types induced decreases in cell viability after 24 hours at a concentration of 100 μg/ml. SPIONs caused the lowest toxicity in HUVECs. By contrast, SiNPs induced pronounced necrosis and apoptosis. A time course experiment showed the gradual toxic effect of all the tested NPs. CNTCOOHs inhibited tetrazolium derivatives at 100 μg/ml, causing false negative results from the WST-8 and LDH assay. In summary, our data demonstrate that the presented “three-in-one” screening assay is capable of evaluating NP toxicity effectively and reliably. Due to its simultaneous utilization of two different methods to assess cell viability, this assay is also capable of revealing, if NPs interfere with tetrazolium salts.

Abstract 3559: Galectin-1 upregulates CXCR4 in glioblastoma through upregulation of CXCR4 via NF-κB activation

Abstract Background: Despite aggressive treatment, including surgical resection, radiation and chemotherapy, over 90% of glioblastoma (GB) patients experience tumor recurrence. This may be due to high migratory potential, abnormal angiogenesis, molecular heterogeneity and a strong immunosuppressive environment. An earlier report from our laboratory demonstrated that GB cells express high levels of carbohydrate-binding galectin protein: galectin-1 (Gal-1). However, the role of galectin-1 in GB remains elusive. The aim of this study was to evaluate whether galectin-1 promotes tumor progression through upregulation of CXCR4 via NF-κB in in vitro GB models. Methods: To investigate the molecular mechanisms of Gal-1 on tumor progression, we used RT-PCR and western blots techniques. Gal-1 was stably over-expressed in the U118 and U87MG cell line. Gal-1 (+/-) sub-clones were assayed for attachment, proliferation, migration, and tube formation. Results: Gal-1 protein expression was assessed in human GB cells. We found increased Gal-1 protein expression in human GB cells and tissue. To investigate the effects of Gal-1 silencing on angiogenesis in vitro, we collected conditioned media from Gal-1-knockdown U118 and U87MG cells then applied this to human umbilical vein endothelial cells (HUVEC) culture to analyze endothelial cell proliferation, migration, and tube formation. Proliferation rate, migrating capability, and tube formation decreased significantly when HUVECs were cultured with concentrated conditioned media from Gal-1-knockdown cells. This data suggests that Gal-1 in GB cells may promote tumor angiogenesis both in vitro and co-culture. Results showed that silencing of Gal-1 in U118 and U87MG cells significantly repressed CXCR4 expression at the mRNA level and protein levels. To further explore the role of CXCR4 in GB cells, CXCR4 knockdown resulted in significant inhibition of cell migration and clonogenic ability in vitro. Thus, Gal-1 might upregulate CXCR4 expression to promote tumor progression. A previous laboratory study indicated that NF-κB promotes cancer cell motility by upregulating CXCR4 expression. Luciferase reporter assay was used to determine the involvement of NF-κB in Gal-1-induced CXCR4 expression in U118 and U87MG cells. NF-κB luciferase activity was significantly decreased in Gal-1-knockdown cells. Moreover, decreased CXCR4 promoter activity and protein expression in Gal-1-knockdown cells were observed. This data indicates that Gal-1 might modulate CXCR4 expression via NF-κB activation. Conclusion: Our in vitro cell culture results suggest that upregulation of galectin-1 and CXCR4 might at least in part explain the aggressive infiltrative biology of GB and the Gal-1-CXCR4 axis may serve as a therapeutic target. Further studies in different animal models are warranted. Citation Format: David Cachia, Rachel Malley, William A. Vandergrift, Sunil Patel, Gabriel A. Rabinovich, Arabinda Das. Galectin-1 upregulates CXCR4 in glioblastoma through upregulation of CXCR4 via NF-κB activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3559.

Maternal hypertension and feto-placental growth restriction is reversed by sildenafil: Evidence of independent effects of circulating nitric oxide levels

Sildenafil has shown nitric oxide (NO)-independent pleiotropic effects, however the mechanisms involved are unclear. We investigated the protective effects of sildenafil against hypertension in pregnancy and feto-placental growth restriction induced by NO inhibition, and if sodium nitrite-derived NO formation influences sildenafil effects. We evaluated the plasmatic levels of NO metabolites, cyclic guanosine monophosphate (cGMP), oxidative stress and myeloperoxidase, which are involved in endothelial dysfunction during hypertension in pregnancy. Also, we performed in vitro experiments to examine cell viability and NO synthesis in human umbilical vein endothelial cells (HUVECs) cultures incubated with plasma from healthy or hypertensive pregnant rats treated (or not) with both drugs, either alone or in association. Sildenafil blunted hypertension in pregnancy and protected against feto-placental growth restriction induced by NO inhibition and these effects of sildenafil alone were similar to those presented by its association with sodium nitrite. Protective effects of sildenafil were observed even with low plasmatic NO levels and were not followed by increases in cGMP levels. Also, sildenafil, but not sodium nitrite, blunted the increases in myeloperoxidase activity. Both drugs (isolated or in association) presented antioxidant effects. Plasma from hypertensive pregnant rats treated with sildenafil, but not sodium nitrite alone, increased the viability of HUVECs. NO synthesis in HUVECs cultures was increased with plasma from rats treated with both drugs. We conclude that sildenafil effects are not dependent of circulating NO levels in hypertension and feto-placental growth restriction. These findings may reflect a protection against myeloperoxidase and pro-oxidant activation in hypertension in pregnancy.

Effects of different hypoxia degrees on endothelial cell cultures—Time course study

IntroductionExposure of the endothelial cells to hypoxia, the decrease in oxygen supply can trigger an endothelial response. This response is involved in inflammatory diseases, tumorigenesis, and also with the micro vascular damage associated with aging.The aim of our study was to determine the hypoxia/re-oxygenation induced response in vitro, using human umbilical vein endothelial cells (HUVEC) cultures, at different time points with focus on cell viability, apoptosis oxidative stress and angiogenesis stimulation. Materials and methodsCells were exposed to 10%, 5% or 0% O2 for 6h, 12h, and 24h. Viability was measured through colorimetry, apoptosis – annexin V-FITC staining, DNA lesions (γH2AX), endothelial activation (sICAM1), angiogenesis (HIF1α), oxidative stress (malondialdehyde, superoxidismutase and NFκB activation) were determined by ELISA, Western Blot and spectrophotometry. Results and discussionHypoxia decreased viability, increased apoptosis, oxidative stress, endothelial activation and angiogenesis, depending on O2 concentration and time exposure. Short exposures to 5% and 10% O2, efficiently activated anti-apoptotic mechanisms through NFκB activation, HIF1α and γH2AX related DNA damage repair pathways. However, severe hypoxia and longer exposures to mild hypoxia induced high oxidative stress related damage and eventually led to apoptosis, through strong increases of HIF1α and accumulating DNA lesions.

Retracted: Anti-ribosomal-phosphoprotein autoantibodies penetrate to neuronal cells via neuronal growth associated protein, affecting neuronal cells in vitro.

OBJECTIVE Anti-ribosomal-phosphoprotein antibodies (anti-Ribos.P Abs) are detected in 10-45% of NPSLE patients. Intracerebroventricular administration of anti-ribosomal-P Abs induces depression-like behaviour in mice. We aimed to discern the mechanism by which anti-Ribos.P Abs induce behavioural changes in mice. METHODS Anti-Ribos.P Abs were exposed to human and rat neuronal cell cultures, as well as to human umbilical vein endothelial cell cultures for a control. The cellular localization of anti-Ribo.P Abs was found by an immunofluorescent technique using a confocal microscope. Identification of the target molecules was undertaken using a cDNA library. Immunohistochemistry and an inhibition assay were carried out to confirm the identity of the target molecules. Neuronal cell proliferation was measured by bromodeoxyuridine, and Akt and Erk expression by immunoblot. RESULTS Human anti-Ribos.P Abs penetrated into human neuronal cells and rat hippocampal cell cultures in vitro, but not to endothelial cells as examined. Screening a high-content human cDNA-library with anti-Ribos.P Abs identified neuronal growth-associated protein (GAP43) as a target for anti-Ribos.P Abs. Ex vivo anti-Ribos.P Abs bind to mouse brain sections of hippocampus, dentate and amygdala. Anti-Ribos.P Abs brain-binding was prevented by GAP43 protein. Interestingly, GAP43 inhibited in a dose-dependent manner the anti-Ribos.P Abs binding to recombinant-ribosomal-P0, indicating mimicry between the ribosomal-P0 protein and GAP43. Furthermore, anti-Ribos.P Abs reduced neuronal cell proliferation activity in vitro (P < 0.001), whereas GAP43 decreased this inhibitory activity by a factor of 7.6. The last was related to Akt and Erk dephosphorylation. CONCLUSION Anti-Ribos.P Abs penetrate neuronal cells in vitro by targeting GAP43. Anti -Ribos.P Abs inhibit neuronal-cell proliferation via inhibition of Akt and Erk. Our data contribute to deciphering the mechanism for anti-Ribos.P Abs' pathogenic activity in NPSLE.

Effect of Human Umbilical Vein Endothelial Cell Culture Media (HUVEC-CM) on of Endothelial Progenitor Cells Differentiation and Construction of Enzyme-Sensitive Hydrogel Culture System

Effect of Human Umbilical Vein Endothelial Cell Culture Media (HUVEC-CM) on of Endothelial Progenitor Cells Differentiation and Construction of Enzyme-Sensitive Hydrogel Culture System

Effects of 17β-estradioland raloxifene on endothelial OPG and RANKL secretion.

This study aims to asses the effects of estradiol vs. raloxifene on the levels of osteoprotegerin and soluble Receptor Activator of Nuclear Factor kB Ligand (sRANKL) in Human Umbilical Vein Endothelial Cells (HUVEC) culture in standard and calcifying medium. Human Umbilical Vein Endothelial Cells were isolated from human umbilical vein by standard method. The supernatant concentrations of osteoprotegerin (OPG) and sRANKL (ELISA) were determined after incubation with glicerophosphate, estradiol , raloxifene, glicerophoshate and estradiol, glicerophosphate and raloxifene in comparison with control group at four designated time points (0, 1, 2 and 4 days of incubation). Incubation of estradiol with HUVEC colony lowered the OPG level significantly after day 2 and 4. Meantime, the level of sRANKL was stable. Raloxifene added to standard growth medium also significantly lowered OPG concentration after day 4 only, with no impact on sRANKL concentration. When added to calcifying medium, both estradiol and raloxifene significantly changed OPG level during the experiment. In all treated groups OPG levels were lower than in groups exposed to calcifying medium only. Neither estradiol, nor raloxifene changed sRANKL levels during the experiment. Estradiol and raloxifene affect OPG secretion from endothelial cells in vitro which may suggest their modifying role in pathogenesis of vascular calcification in postmenopausal women.

Inhibition of Myeloperoxidase- and Neutrophil-Mediated Hypochlorous Acid Formation in Vitro and Endothelial Cell Injury by (-)-Epigallocatechin Gallate.

Myeloperoxidase (MPO) plays important roles in various diseases through its unique chlorinating activity to catalyze excess hypochlorous acid (HOCl) formation. Epidemiological studies indicate an inverse correlation between plant polyphenol consumption and the incidence of cardiovascular diseases. Here we showed that (-)-epigallocatechin gallate (EGCG), the main flavonoid present in green tea, dose-dependently inhibited MPO-mediated HOCl formation in vitro (chlorinating activities of MPO: 50.2 ± 5.7% for 20 μM EGCG versus 100 ± 5.6% for control, P < 0.01). UV-vis spectral and docking studies indicated that EGCG bound to the active site (heme) of MPO and resulted in the accumulation of compound II, which was unable to produce HOCl. This flavonoid also effectively inhibited HOCl generation in activated neutrophils (HOCl formation: 65.0 ± 5.6% for 20 μM EGCG versus 100 ± 6.2% for control, P < 0.01) without influencing MPO and Nox2 release and superoxide formation, suggesting that EGCG specifically inhibited MPO but not NADPH oxidase activity in activated neutrophils. Moreover, EGCG inhibited MPO (or neutrophil)-mediated HOCl formation in human umbilical vein endothelial cells (HUVEC) culture and accordingly protected HUVEC from MPO (or neutrophil)-induced injury (P < 0.05, all cases), although it did not induce cytotoxicity to HUVEC (P > 0.05, all cases). Our results indicate that dietary EGCG is an effective and specific inhibitor of MPO activity and may participate in the regulation of immune responses at inflammatory sites.

HDL-associated ApoM is anti-apoptotic by delivering sphingosine 1-phosphate to S1P1 & S1P3 receptors on vascular endothelium

BackgroundHigh-density Lipoprotein (HDL) attenuates endothelial cell apoptosis induced by different cell-death stimuli such as oxidation or growth factor deprivation. HDL is the main plasma carrier of the bioactive lipid sphingosine 1-phosphate (S1P), which it is a signaling molecule that promotes cell survival in response to several apoptotic stimuli. In HDL, S1P is bound to Apolipoprotein M (ApoM), a Lipocalin that is only present in around 5% of the HDL particles. The goal of this study is to characterize ApoM-bound S1P role in endothelial apoptosis protection and the signaling pathways involved.MethodsHuman umbilical vein endothelial cells (HUVEC) cultures were switched to serum/grow factor deprivation medium to induce apoptosis and the effect caused by the addition of ApoM and S1P analyzed.ResultsThe addition of HDL+ApoM or recombinant ApoM-bound S1P promoted cell viability and blocked apoptosis, whereas HDL-ApoM had no protective effect. Remarkably, S1P exerted a more potent anti-apoptotic effect when carried by ApoM as compared to albumin, or when added as free molecule. Mechanistically, cooperation between S1P1 and S1P3 was required for the HDL/ApoM/S1P-mediated anti-apoptotic ability. Furthermore, AKT and ERK phosphorylation was also necessary to achieve the anti-apoptotic effect of the HDL/ApoM/S1P complex.ConclusionsAltogether, our results indicate that ApoM and S1P are key elements of the anti-apoptotic activity of HDL and promote optimal endothelial function.

In vitro toxicity of FemOn, FemOn-SiO2 composite, and SiO2-FemOn core-shell magnetic nanoparticles.

Over the last decade, magnetic iron oxide nanoparticles (IONPs) have drawn much attention for their potential biomedical applications. However, serious in vitro and in vivo safety concerns continue to exist. In this study, the effects of uncoated, FemOn-SiO2 composite flake-like, and SiO2-FemOn core-shell IONPs on cell viability, function, and morphology were tested 48 h postincubation in human umbilical vein endothelial cell culture. Cell viability and apoptosis/necrosis rate were determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and annexin V-phycoerythrin kit, respectively. Cell morphology was evaluated using bright-field microscopy and forward and lateral light scattering profiles obtained with flow cytometry analysis. All tested IONP types were used at three different doses, that is, 0.7, 7.0, and 70.0 μg. Dose-dependent changes in cell morphology, viability, and apoptosis rate were shown. At higher doses, all types of IONPs caused formation of binucleated cells suggesting impaired cytokinesis. FemOn-SiO2 composite flake-like and SiO2-FemOn core-shell IONPs were characterized by similar profile of cytotoxicity, whereas bare IONPs were shown to be less toxic. The presence of either silica core or silica nanoflakes in composite IONPs can promote cytotoxic effects.