In vitro blood-brain barrier (BBB) models from primary brain endothelial cells can closely resemble the in vivo BBB, offering valuable models to assay BBB functions and to screen potential central nervous system drugs. We have recently developed an in vitro BBB model using primary porcine brain endothelial cells. The model shows expression of tight junction proteins and high transendothelial electrical resistance, evidence for a restrictive paracellular pathway. Validation studies using small drug-like compounds demonstrated functional uptake and efflux transporters, showing the suitability of the model to assay drug permeability. However, one limitation of in vitro model permeability measurement is the presence of the aqueous boundary layer (ABL) resulting from inefficient stirring during the permeability assay. The ABL can be a rate-limiting step in permeation, particularly for lipophilic compounds, causing underestimation of the permeability. If the ABL effect is ignored, the permeability measured in vitro will not reflect the permeability in vivo. To address the issue, we explored the combination of in vitro permeability measurement using our porcine model with the pKa(FLUX) method in pCEL-X software to correct for the ABL effect and allow a detailed analysis of in vitro (transendothelial) permeability data, Papp. Published Papp using porcine models generated by our group and other groups are also analyzed. From the Papp, intrinsic transcellular permeability (P0) is derived by simultaneous refinement using a weighted nonlinear regression, taking into account permeability through the ABL, paracellular permeability and filter restrictions on permeation. The in vitro P0 derived for 22 compounds (35 measurements) showed good correlation with P0 derived from in situ brain perfusion data (r(2)=0.61). The analysis also gave evidence for carrier-mediated uptake of naloxone, propranolol and vinblastine. The combination of the in vitro porcine model and the software analysis provides a useful tool to better predict BBB permeability in vivo and gain better mechanistic information about BBB permeation.
Ardisia crispa Thunb. A. DC. (Primulaceae) has been used extensively as folk-lore medicine in South East Asia including China and Japan to treat various inflammatory related diseases. Ardisia crispa root hexane fraction (ACRH) has been thoroughly studied by our group and it has been shown to exhibit anti-inflammatory, anti-hyperalgesic, anti-arthritic, anti-ulcer, chemoprevention and suppression against inflammation-induced angiogenesis in various animal model. Nevertheless, its effect against human endothelial cells in vitro has not been reported yet. Hence, the aim of the study is to investigate the potential antiangiogenic property of ACRH in human umbilical vein endothelial cells (HUVECs) and zebrafish embryo model. ACRH was separated from the crude ethanolic extract of the plant's root in prior to experimental studies. MTT assay revealed that ACRH exerted a concentration-dependent antiproliferative effect on HUVEC with the IC50 of 2.49 ± 0.04 μg/mL. At higher concentration (10 μg/mL), apoptosis was induced without affecting the cell cycle distribution. Angiogenic properties including migration, invasion and differentiation of HUVECs, evaluated via wound healing, trans-well invasion and tube formation assay respectively, were significantly suppressed by ACRH in a concentration-dependent manner. Noteworthily, significant antiangiogenic effects were observed even at the lowest concentration used (0.1 μg/mL). Expression of proMMP-2, vascular endothelial growth factor (VEGF)-C, VEGF-D, Angiopoietin-2, fibroblast growth factor (FGF)-1, FGF-2, Follistatin, and hepatocyte growth factor (HGF) were significantly reduced in various degrees by ACRH. The ISV formation in zebrafish embryo was significantly suppressed by ACRH at the concentration of 5 μg/mL. These findings revealed the potential of ACRH as antiangiogenic agent by suppressing multiple proangiogenic proteins. Thus, it can be further verified via the transcription of these proteins from their respective DNA, in elucidating their exact pathways.
Matched MeSH terms: Human Umbilical Vein Endothelial Cells/metabolism*; Human Umbilical Vein Endothelial Cells/pathology
Curcuma zedoaria also known as Temu putih is traditionally used in food preparations and treatment of various ailments including cancer. The cytotoxic activity of hexane, dichloromethane, ethyl acetate, methanol, and the methanol-soxhlet extracts of Curcuma zedoaria rhizomes was tested on two human cancer cell lines (Ca Ski and MCF-7) and a noncancer cell line (HUVEC) using MTT assay. Investigation on the chemical components in the hexane and dichloromethane fractions gave 19 compounds, namely, labda-8(17),12 diene-15,16 dial (1), dehydrocurdione (2), curcumenone (3), comosone II (4), curcumenol (5), procurcumenol (6), germacrone (7), zerumbone epoxide (8), zederone (9), 9-isopropylidene-2,6-dimethyl-11-oxatricyclo[6.2.1.0(1,5)]undec-6-en-8-ol (10), furanodiene (11), germacrone-4,5-epoxide (12), calcaratarin A (13), isoprocurcumenol (14), germacrone-1,10-epoxide (15), zerumin A (16), curcumanolide A (17), curcuzedoalide (18), and gweicurculactone (19). Compounds (1-19) were evaluated for their antiproliferative effect using MTT assay against four cancer cell lines (Ca Ski, MCF-7, PC-3, and HT-29). Curcumenone (3) and curcumenol (5) displayed strong antiproliferative activity (IC50 = 8.3 ± 1.0 and 9.3 ± 0.3 μg/mL, resp.) and were found to induce apoptotic cell death on MCF-7 cells using phase contrast and Hoechst 33342/PI double-staining assay. Thus, the present study provides basis for the ethnomedical application of Curcuma zedoaria in the treatment of breast cancer.
Matched MeSH terms: Human Umbilical Vein Endothelial Cells
Syzygium campanulatum Korth (Myrtaceae) is an evergreen shrub rich in phenolics, flavonoid antioxidants, and betulinic acid. This study sought to investigate antiangiogenic and anti-colon cancer effects of S.C. standardized methanolic extract.
Matched MeSH terms: Human Umbilical Vein Endothelial Cells/cytology; Human Umbilical Vein Endothelial Cells/enzymology; Human Umbilical Vein Endothelial Cells/metabolism
Interferon-gamma (IFN-γ) is a pleiotropic cytokine that plays a critical role in mediating an array of immune responses including promotes antiviral activity, facilitates macrophage activation, controls Th1/Th2 balance, and regulates cellular apoptosis and proliferation. A few articles have previously reviewed the effects of IFN-γ in the regulation of barrier permeability, but none of these articles focuses on barrier function of endothelial cells. This review aims to discuss the regulatory mechanisms of IFN-γ on endothelial barrier function and its underlying signaling pathways. Articles were retrieved from electronic databases such as PubMed and Google Scholar using keywords "Interferon-gamma", "endothelial cells", "barrier function", and "signaling pathway". The articles published between 2000 and 2022 that are related to the aforementioned topics were selected. A few journals published beyond this period were also included due to limited information available. The results showed that IFN-γ modulates endothelial barrier function, mainly involves small GTPases, STAT1-dependent pathway, p38 MAPK and nitric oxide. In conclusion, more in depth cellular and molecular studies are needed to elucidate the pathways of IFN-γ in the regulation of endothelial barrier function.
Nitric oxide (NO), produced by endothelial nitric oxide synthase (eNOS), is a major antiatherogenic factor in the blood vessel. Oxidative stress plays an important role in the pathogenesis of various cardiovascular diseases, including atherosclerosis. Decreased availability of endothelial NO promotes the progression of endothelial dysfunction and atherosclerosis. Rutin is a flavonoid with multiple cardiovascular protective effects. This study aimed to investigate the effects of rutin on eNOS and NO production in cultured human umbilical vein endothelial cells (HUVEC). HUVEC were divided into four groups: control; oxidative stress induction with 180 μM H₂O₂; treatment with 300 μM rutin; and concomitant induction with rutin and H₂O₂ for 24 hours. HUVEC treated with rutin produced higher amount of NO compared to control (P < 0.01). In the oxidative stress-induced HUVEC, rutin successfully induced cells' NO production (P < 0.01). Rutin promoted NO production in HUVEC by inducing eNOS gene expression (P < 0.05), eNOS protein synthesis (P < 0.01), and eNOS activity (P < 0.05). Treatment with rutin also led to increased gene and protein expression of basic fibroblast growth factor (bFGF) in HUVEC. Therefore, upregulation of eNOS expression by rutin may be mediated by bFGF. The results showed that rutin may improve endothelial function by augmenting NO production in human endothelial cells.
Matched MeSH terms: Human Umbilical Vein Endothelial Cells/drug effects*; Human Umbilical Vein Endothelial Cells/metabolism*
Activation of inflammatory pathways via reactive oxygen species (ROS) by free fatty acids (FFA) in obesity gives rise to insulin resistance and endothelial dysfunction. Withaferin A (WA), possesses both antioxidant and anti-inflammatory properties and therefore would be a good strategy to suppress palmitic acid (PA)-induced oxidative stress and inflammation and hence, insulin resistance and dysfunction in the endothelium. Effect of WA on PA-induced insulin resistance in human umbilical vein endothelial cells (HUVECs) was determined by evaluating insulin signaling mechanisms whilst effect of this drug on PA-induced endothelial dysfunction was determined in acetylcholine-mediated relaxation in isolated rat aortic preparations. WA significantly inhibited ROS production and inflammation induced by PA. Furthermore, WA significantly decreased TNF-α and IL-6 production in endothelial cells by specifically suppressing IKKβ/NF-κβ phosphorylation. WA inhibited inflammation-stimulated IRS-1 serine phosphorylation and improved the impaired insulin PI3-K signaling, and restored the decreased nitric oxide (NO) production triggered by PA. WA also decreased endothelin-1 and plasminogen activator inhibitor type-1 levels, and restored the impaired endothelium-mediated vasodilation in isolated aortic preparations. These findings suggest that WA inhibited both ROS production and inflammation to restore impaired insulin resistance in cultured endothelial cells and improve endothelial dysfunction in rat aortic rings.
Matched MeSH terms: Human Umbilical Vein Endothelial Cells
Parkia speciosa Hassk is a traditional medicinal plant with strong antioxidant and hypoglycemic properties. This study aims to investigate the total phenolic content, antioxidant, cytotoxic and antiangiogenic effect of eight extracts from P. speciosa empty pods. The extracts were found to contain high levels of total phenols and demonstrated strong antioxidant effect in DPPH scavenging test. In rat aortic rings, P. speciosa extracts significantly inhibited the microvessel outgrowth from aortic tissue explants by more than 50%. The antiangiogenic activity was further confirmed by tube formation on matrigel matrix involving human endothelial cells. Cytotoxic effect was evaluated by XTT test on endothelial cells as a model of angiogenesis versus a panel of human cancer and normal cell lines. Basically the extracts did not show acute cytotoxicity. Morphology examination of endothelial cells indicated induction of autophagy characterized by formation of plenty of cytoplasmic vacuoles. The extracts were found to work by decreasing expression of vascular endothelial growth factor in endothelial cells.
This study evaluated the in vitro antioxidant capacities of extracts from Pleurotus pulmonarius via Folin-Ciocalteu, 1,1-diphenyl-2-picrylhydrazyl free radical scavenging, metal chelating, cupric ion reducing antioxidant capacity, and lipid peroxidation inhibition assays. Extract compositions were determined by phenol-sulfuric acid; Coomassie Plus (Bradford) protein; Spectroquant zinc, copper, and manganese test assays; and liquid chromatography-tandem mass spectrometry (LC/MS/MS) and gas chromatography-mass spectrometry (GC/MS). Methanol-dichloromethane extract, water fraction, hot water, aqueous extract and hexane fraction exhibited the most potent extracts in the antioxidant activities. LC/MS/MS and GC/MS showed that the extracts contained ergothioneine, ergosterol, flavonoid, and phenolic compounds. The selected potent extracts were evaluated for their inhibitory effect against oxidation of human low-density lipoproteins and protective effects against hydrogen peroxide-induced cytotoxic injury in human aortic endothelial cells. The crude aqueous extract was deemed most potent for the prevention of human low-density lipoprotein oxidation and endothelial membrane damage. Ergothioneine might be the compound responsible for the activities, as supported by previous reports. Thus, P. pulmonarius may be a valuable antioxidant ingredient in functional foods or nutraceuticals.
This study evaluates the in vitro inhibition of angiotensin-converting enzyme (ACE) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA) by Pleurotus pulmonarius extracts. The protective effect on the endothelial membrane against oxidative stress through the protection of nitric oxide bioavailability, as well as inhibition of endocan expression, was evaluated using human aortic endothelial cells (HAECs). Crude cold aqueous extract exhibited the most potent inhibitory activities against ACE and HMG-CoA reductase, with 61.79% and 44.30% inhibition, respectively. It also protected the bioavailability of NO released by HAECs, with 84.88% cell viability. The crude hot water extract was the most potent in inhibiting endocan expression, with 18.61% inhibition.
Numerous protocols for the isolation of bovine aortic endothelial cells have been described in the previous literature. However, these protocols prevent researchers from obtaining the pure population of endothelial cells. Thus, this study aimed to develop a new and economical method for the isolation of pure endothelial cells by introducing a new strategy to the enzymatic digestion method proposed by previous researchers.
miRNAs are important regulators of cellular senescence yet the extent of their involvement remains to be investigated. We sought to identify miRNAs that are involved in cytokine-induced premature senescence (CIPS) in endothelial cells. CIPS was established in young human pulmonary microvascular endothelial cells (HMVEC-Ls) following treatment with a sublethal dose (20ng/ml) of tumor necrosis factor alpha (TNF-α) for 15days. In parallel, HMVEC-Ls were grown and routinely passaged until the onset of replicative senescence (RS). Differential expression analysis following miRNA microarray profiling revealed an overlapped of eight deregulated miRNAs in both the miRNA profiles of RS and TNF-α-induced premature senescence cells. Amongst the deregulated miRNAs were members of the miR 17-92 cluster which are known regulators of angiogenesis. The role of hsa-miR-20b in TNF-α-induced premature senescence, a paralog member of the miR 17-92 cluster, was further investigated. Biotin-labeled hsa-miR-20b captured the enriched transcripts of retinoblastoma-like 1 (RBL1), indicating that RBL1 is a target of hsa-miR-20b. Knockdown of hsa-miR-20b attenuated premature senescence in the TNF-α-treated HMVEC-Ls as evidenced by increased cell proliferation, increased RBL1 mRNA expression level but decreased protein expression of p16INK4a, a cellular senescence marker. These findings provide an early insight into the role of hsa-miR-20b in endothelial senescence.
Diabetic retinopathy (DR), one of the leading causes of visual impairment and blindness worldwide, is one of the major microvascular complications in diabetes mellitus (DM). Globally, DR prevalence among DM patients is 25%, and 6% have vision-threatening problems among them. With the higher incidence of DM globally, more DR cases are expected to be seen in the future. In order to comprehend the pathophysiological mechanism of DR in humans and discover potential novel substances for the treatment of DR, investigations are typically conducted using various experimental models. Among the experimental models, in vivo models have contributed significantly to understanding DR pathogenesis. There are several types of in vivo models for DR research, which include chemical-induced, surgical-induced, diet-induced, and genetic models. Similarly, for the in vitro models, there are several cell types that are utilised in DR research, such as retinal endothelial cells, Müller cells, and glial cells. With the advancement of DR research, it is essential to have a comprehensive update on the various experimental models utilised to mimic DR environment. This review provides the update on the in vitro, in vivo, and ex vivo models used in DR research, focusing on their features, advantages, and limitations.
The present study aimed to investigate the mechanisms underlying the anti-inflammatory and anti-angiogenic effects of ethyl-p-methoxycinnamate isolated from Kaempferia galanga.
Matched MeSH terms: Human Umbilical Vein Endothelial Cells/drug effects
Glioblastoma multiforme (GBM) is one of the most common primary brain tumours in adults, accounting for almost 65% of all cases. Among solid tumours, GBM is characterised by strong angiogenesis, including the highest degree of vascular proliferation and endothelial cell hyperplasia. Despite numerous improvements in existing treatment approaches, the prognosis of GBM patients remains poor, with a mean survival of only 14.6 months. Growing evidence has shown significant overexpression of the ephrin type-A receptor 2 (EphA2) receptor in various malignancies, including GBM, as well as a correlation to poor prognoses. It is believed that EphA2 receptors play important roles in mediating GBM tumourigenesis, including invasion, metastasis, and angiogenesis. Despite the clinical and pathological importance of tumour-associated vasculature, the underlying mechanism involving EphA2 is poorly known. Here, we have summarised the current knowledge in the field regarding EphA2 receptors' roles in the angiogenesis of GBM.
2,6-bis-(4-hydroxyl-3-methoxybenzylidine)cyclohexanone (BHMC) has been proven to selectively inhibit the synthesis of proinflammatory mediators in lipopolysaccharide-induced U937 monocytes through specific interruption of p38 Mitogen-Activated Protein Kinase enzymatic activity and improves the survival rate in a murine lethal sepsis model. The present study addressed the effects of BHMC upon lipopolysaccharide-induced endothelial dysfunction in human umbilical vein endothelial cells to determine the underlying mechanisms. The cytotoxicity effect of BHMC on HUVEC were determined by MTT assay. The effects of BHMC on endothelial dysfunction induced by lipopolysaccharide such as endothelial hyperpermeability, monocyte-endothelial adhesion, transendothelial migration, up-regulation of adhesion molecules and chemokines were evaluated. The effects of BHMC at transcriptional and post-translational levels were determined by Reverse Transcriptase-Polymerase Chain Reaction and Western Blots. The mode of action of BHMC was dissected by looking into the activation of Nuclear Factor-kappa B and Mitogen-Activated Protein Kinases. BHMC concentration-dependently reduced endothelial hyperpermeability, leukocyte-endothelial cell adhesion and monocyte transendothelial migration through inhibition of the protein expression of adhesion molecules (Intercellular Adhesion Molecule-1 and Vascular Cell Adhesion Molecule-1) and secretion of chemokines (Monocyte Chemotactic Protein-1) at the transcriptional level. BHMC restored endothelial dysfunction via selective inhibition of p38 Mitogen-Activated Protein Kinase enzymatic activity which indirectly prevents the activation of Nuclear Factor-kappaB and Activator Protein-1 transcription factors. These findings further support earlier observations on the inhibition of BHMC on inflammatory events through specific disruption of p38 Mitogen-Activated Protein Kinase enzymatic activity and provide new insights into the inhibitory effects of BHMC on lipopolysaccharide-induced endothelial dysfunction.
Matched MeSH terms: Human Umbilical Vein Endothelial Cells/drug effects*; Human Umbilical Vein Endothelial Cells/metabolism
Histamine is established as a potent inflammatory mediator and it is known to increased endothelial permeability by promoting gap formation between endothelial cells. Previous studies have shown that aqueous extract of Bixa orellana leaves (AEBO) exhibits antihistamine activity in vivo, yet the mechanism of its action on endothelial barrier function remains unclear. Therefore, the current study aimed to determine the protective effect of AEBO against histamine-induced hyperpermeability in vitro.
Matched MeSH terms: Human Umbilical Vein Endothelial Cells/drug effects
The endothelial barrier function is tightly controlled by a broad range of signaling cascades including nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathway. It has been proposed that disturbances in NO and cGMP production could interfere with proper endothelial barrier function. In this study, we assessed the effect of interferon-gamma (IFN-gamma), a pro-inflammatory cytokine, on NO and cGMP levels and examined the mechanisms by which NO and cGMP regulate the IFN-gamma-mediated HUVECs hyperpermeability. The flux of fluorescein isothiocyanate-labeled dextran across cell monolayers was used to study the permeability of endothelial cells. Here, we found that IFN-gamma significantly attenuated basal NO concentration and the increased NO levels supplied by a NO donor, sodium nitroprusside (SNP). Besides, application of IFN-gamma also significantly attenuated both the basal cGMP concentration and the increased cGMP production donated by a cell permeable cGMP analogue, 8-bromo-cyclic GMP (8-Br-cGMP). In addition, exposure of the cell monolayer to IFN-gamma significantly increased HUVECs basal permeability. However, L-NAME pretreatment did not suppress IFN-gamma-induced HUVECs hyperpermeability. L-NAME pretreatment followed by SNP or SNP pretreatment partially reduced IFN-gamma-induced HUVECs hyperpermeability. Pretreatment with a guanylate cyclase inhibitor, 6-anilino-5,8-quinolinedione (LY83583), led to a further increase in IFN-gamma-induced HUVECs hyperpermeability. The findings suggest that the mechanism underlying IFN-gamma-induced increased HUVECs permeability is partly related to the inhibition of NO production.
Matched MeSH terms: Human Umbilical Vein Endothelial Cells/drug effects*; Human Umbilical Vein Endothelial Cells/metabolism*
Endothelial barrier dysfunction leads to increased endothelial permeability and is an early step in the development of vascular inflammatory diseases such as atherosclerosis. Interferon-γ (IFN-γ), a proinflammatory cytokine, is known to cause increased endothelial permeability. However, the mechanisms by which IFN-γ disrupts the endothelial barrier have not been clarified. This study aimed to investigate how IFN-γ impairs the endothelial barrier integrity by specifically examining the roles of caldesmon, adherens junctions (AJs) and p38 mitogen-activated protein (MAP) kinase in IFN-γ-induced endothelial barrier dysfunction. IFN-γ exhibited a biphasic effect on caldesmon localization and both the structural organization and protein expression of AJs. In the early phase (4-8 h), IFN-γ induced the formation of peripheral caldesmon bands and discontinuous AJs, while AJ protein expression was unchanged. Interestingly, IFN-γ also stimulated caldesmon phosphorylation, resulting in actin dissociation from caldesmon at 8 h. Conversely, changes seen in the late phase (16-24 h) included cytoplasmic caldesmon dispersal, AJ linearization and junctional area reduction, which were associated with reduced membrane, cytoskeletal and total AJ protein expression. In addition, IFN-γ enhanced myosin binding to caldesmon at 12 h and persisted up to 24 h. Furthermore, inhibition of p38 MAP kinase by SB203580 did not reverse either the early or late phase changes observed. These data suggest that IFN-γ may activate signaling molecules other than p38 MAP kinase. In conclusion, our findings enhance the current understanding of how IFN-γ disrupts endothelial barrier function and reveal potential therapeutic targets, such as caldesmon and AJs, for the treatment of IFN-γ-associated vascular inflammatory diseases.
Matched MeSH terms: Endothelial Cells/metabolism; Human Umbilical Vein Endothelial Cells
Endothelial hyperpermeability represents an initiating step in early atherosclerosis and it often occurs as a result of endothelial barrier dysfunction. Asiatic acid, a major triterpene isolated from Centella asiatica (L.) Urban, has previously been demonstrated to protect against tumor necrosis factor (TNF)-α-induced endothelial barrier dysfunction. The present study aimed to investigate the mechanisms underlying the barrier protective effect of asiatic acid in human aortic endothelial cells (HAECs). The localization of F-actin, diphosphorylated myosin light chain (diphospho-MLC), adherens junctions (AJs) and tight junctions (TJs) was studied using immunocytochemistry techniques and confocal microscopy. Their total protein expressions were examined using western blot analysis. The endothelial permeability was assessed using In Vitro Vascular Permeability Assay kits. In addition, intracellular redistribution of the junctional proteins was evaluated using subcellular fractionation kits. We show that asiatic acid stabilized F-actin and diphospho-MLC at the cell periphery and prevented their rearrangement stimulated by TNF-α. However, asiatic acid failed to attenuate cytochalasin D-induced increased permeability. Besides, asiatic acid abrogated TNF-α-induced structural reorganization of vascular endothelial (VE)-cadherin and β-catenin by preserving their reticulum structures at cell-cell contact areas. In addition, asiatic acid also inhibited TNF-α-induced redistribution of occludin and zona occludens (ZO)-1 in different subcellular fractions. In conclusion, the barrier-stabilizing effect of asiatic acid might be associated with preservation of AJs and prevention of TJ redistribution caused by TNF-α. This study provides evidence to support the potential use of asiatic acid in the prevention of early atherosclerosis, which is initiated by endothelial barrier dysfunction.