Umbilical Vein Endothelial Cells Research Articles

Liver‐on‐a‐Chip: In Vitro Human Liver Model of Nonalcoholic Steatohepatitis by Coculturing Hepatocytes, Endothelial Cells, and Kupffer Cells (Adv. Healthcare Mater. 24/2019)

In article number 1901379, Junmin Lee, Ali Khademhosseini, and co-workers, develop an in vitro human liver model of non-alcoholic fatty liver disease (NAFLD) by co-culturing human hepatocytes, umbilical vein endothelial cells (HUVECs), and Kupffer cells (KCs) into spheroids. The proposed system is an appropriate model to mimic and monitor the progression of NAFLD from steatosis into steatohepatitis.

In Vitro Human Liver Model of Nonalcoholic Steatohepatitis by Coculturing Hepatocytes, Endothelial Cells, and Kupffer Cells.

The liver has a complex and unique microenvironment with multiple cell-cell interactions and internal vascular networks. Although nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease with multiple phases, no proper model could fully recapitulate the in vivo microenvironment to understand NAFLD progression. Here, an in vitro human liver model of NAFLD by coculturing human hepatocytes, umbilical vein endothelial cells (HUVECs), and Kupffer cells (KCs) into spheroids is presented. Analysis of indirect cross-talk using conditioned media between steatotic spheroids-composed of hepatocellular carcinoma-derived cells (HepG2) and HUVECs-and mouse KCs reveals that the latter can be activated showing increased cell area, elevated production of reactive oxygen species (ROS), and proinflammatory cytokines. Spheroids incorporating human KCs (HKCs) can also be induced into steatotic stage by supplementing fat. Steatotic spheroids with/without HKCs show different levels of steatotic stages through lipid accumulation and ROS production. Steatotic spheroids made from an immortalized hepatic progenitor cell line (HepaRG) compared to those made from HepG2 cells display similar trends of functionality, but elevated levels of proinflammatory cytokines, and improved reversibility of steatosis. The in vitro human liver system proposed makes strides in developing a model to mimic and monitor the progression of NAFLD.

Atheroprotective effects of methotrexate via the inhibition of YAP/TAZ under disturbed flow

BackgroundAtherosclerosis preferentially develops in regions of disturbed flow (DF). Emerging evidence indicates that yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), which are both effectors of the Hippo pathway, sense different blood flow patterns and regulate atherosclerotic lesions. We previously found that methotrexate (MTX) reduces in-stent neoatherosclerosis, decreases the plaque burden, and has an effect on local fluid shear stress. Here, we investigated the atheroprotective effect of MTX under DF and the mechanisms underlying these properties.MethodsHuman umbilical vein endothelial cells (HUVECs) were subjected to biomechanical stretch using a parallel-plate flow system and treated with or without MTX at therapeutically relevant concentrations. Additionally, an extravascular device was used to induce DF in the left common carotid artery of C57BL/6 mice, followed by treatment with MTX or 0.9% saline. The artery was then assessed histopathologically after 4 weeks on a Western diet.ResultsWe observed that MTX significantly inhibited DF-induced endothelial YAP/TAZ activation. Furthermore, it markedly decreased pro-inflammatory factor secretion and monocyte adhesion in HUVECs but had no effect on apoptosis. Mechanistically, AMPKa1 depletion attenuated these effects of MTX. Accordingly, MTX decreased DF-induced plaque formation, which was accompanied by YAP/TAZ downregulation in vivo.ConclusionsTaken together, we conclude that MTX exerts protective effects via the AMP-dependent kinase (AMPK)-YAP/TAZ pathway. These results provide a basis for the prevention and treatment of atherosclerosis via the inhibition of YAP/TAZ.

Knockdown of TNFAIP1 prevents di-(2-ethylhexyl) phthalate-induced neurotoxicity by activating CREB pathway.

Di-(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer. It has neurotoxicity and exposure to it causes impairment of neurodevelopment, behavior and cognition. However, the molecular mechanisms responsible for the DEHP-induced neurotoxicity are not yet clearly defined. Tumor necrosis factor-induced protein 1 (TNFAIP1) was first discovered in umbilical vein endothelial cells and was further found to be important in the progress of Alzheimer's disease. Herein we explore the mechanism of TNFAIP1 in DEHP-induced neurotoxicity with the involvement of cyclic AMP response elements binding protein (CREB) signaling pathway in a mouse neuroblastoma cell line (N2a cells). We found that exposure to DEHP induced apoptosis and downregulated the expression of brain-derived neurotrophic factor (BDNF), synaptic proteins PSD 95 and synapsin-1 while upregulated the expression of TNFAIP1 and decreased the levels of phosphorylated Akt, CaMK Ⅳ, catalytic subunits of PKA and CREB in CREB signaling pathway. Knockdown of TNFAIP1 using TNFAIP1 small interfering RNA (siRNA) expression vector prevented DEHP from inhibiting CREB pathway, thus reduced apoptosis and restored expression of BDNF, PSD 95 and synapsin-1. Our data indicate that downregulation of TNFAIP1 prevents DEHP-induced neurotoxicity via activating CREB pathway. Therefore, TNFAIP1 is a potential target for relieving the DEHP-induced neurotoxicity and related neurological disorders.

Hypoxia influences the effects of magnesium degradation products on the interactions between endothelial and mesenchymal stem cells

Biodegradable materials like well-documented Magnesium (Mg) are promising for their biocompatibility and tissue regeneration. Since Mg degradation is reported to be oxygen related, the effects of Mg were hypothesised to be influenced by oxygen. As two vital components of bone marrow, endothelial cells (EC) and mesenchymal stem cells (MSC), their interactions represent high scientific interest for tissue engineering and biodegradable Mg application. Human umbilical cord perivascular (HUCPV) and umbilical vein endothelial cell (HUVEC) were selected as sources of MSC and EC, respectively. Two types of coculture models were established to represent different phases of MSC-EC interaction: (i) where cells were physically separated thanks to a transwell and (ii) where cells were allowed to have heterotypic cellular contacts. Cell migration, gene, cytokines, and proliferation were investigated in HUCPV-HUVEC coculture using DNA, flow cytometry, wound healing assay, semi-quantitative real-time polymerase chain reaction (qRT-PCR), and enzyme-linked immunosorbent assay (ELISA). Mg degradation products increased HUCPV migration in transwell under hypoxia. Oxygen tension changed the gene regulation of migratory, angiogenetic or osteogenic regulators. Under contacting coculture and hypoxia, Mg degradation products remarkably increased cytokines (e.g., c-c motif chemokine ligand 2 and vascular endothelial growth factor) and MSC mineralisation. Mg degradation products decreased and increased the MSC proliferation in transwell and in heterotypic-contact coculture, respectively. In summary, this study indicates the roles of low oxygen and heterotypic contact to effects of Mg materials facilitating HUVEC and HUCPV. Statement of significance•Based on the formerly reported monoculture of endothelial cells, mesenchymal stem cell and endothelial cell as two vital components of bone marrow were cocultured and investigated in magnesium degradation products.•The effects of hypoxia and normoxia were compared to shed light on the significance of local environmental oxygen on magnesium biomaterial-based tissue regeneration.•Besides the beneficial effects of magnesium degradation products, the results of two coculture models suggest MSC-EC heterotypic contact can play as promising “downstream approach” for tissue engineering or cell therapy.

KRIT1 Deficiency Promotes Aortic Endothelial Dysfunction.

Loss-of-function mutations of the gene encoding Krev interaction trapped protein 1 (KRIT1) are associated with the pathogenesis of Cerebral Cavernous Malformation (CCM), a major cerebrovascular disease characterized by abnormally enlarged and leaky capillaries and affecting 0.5% of the human population. However, growing evidence demonstrates that KRIT1 is implicated in the modulation of major redox-sensitive signaling pathways and mechanisms involved in adaptive responses to oxidative stress and inflammation, suggesting that its loss-of-function mutations may have pathological effects not limited to CCM disease. The aim of this study was to address whether KRIT1 loss-of-function predisposes to the development of pathological conditions associated with enhanced endothelial cell susceptibility to oxidative stress and inflammation, such as arterial endothelial dysfunction (ED) and atherosclerosis. Silencing of KRIT1 in human aortic endothelial cells (HAECs), coronary artery endothelial cells (HCAECs), and umbilical vein endothelial cells (HUVECs) resulted in increased expression of endothelial proinflammatory adhesion molecules vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1 (ICAM-1) and in enhanced susceptibility to tumor necrosis factor alpha (TNF-α)-induced apoptosis. These effects were associated with a downregulation of Notch1 activation that could be rescued by antioxidant treatment, suggesting that they are consequent to altered intracellular redox homeostasis induced by KRIT1 loss-of-function. Furthermore, analysis of the aorta of heterozygous KRIT1+/− mice fed a high-fructose diet to induce systemic oxidative stress and inflammation demonstrated a 1.6-fold increased expression of VCAM-1 and an approximately 2-fold enhanced fat accumulation (7.5% vs 3.6%) in atherosclerosis-prone regions, including the aortic arch and aortic root, as compared to corresponding wild-type littermates. In conclusion, we found that KRIT1 deficiency promotes ED, suggesting that, besides CCM, KRIT1 may be implicated in genetic susceptibility to the development of atherosclerotic lesions.

Uric acid regulates NLRP3/IL-1\u03b2 signaling pathway and further induces vascular endothelial cells injury in early CKD through ROS activation and K+ efflux

BackgroundChronic kidney disease (CKD) has been considered as a major health problem in the world. Increasing uric acid (UA) could induce vascular endothelial injury, which is closely related to microinflammation, oxidative stress, and disorders of lipids metabolism. However, the specific mechanism that UA induces vascular endothelial cells injury in early CKD remains unknown.MethodsHuman umbilical vein endothelial cells (HUVECs) were cultured and subjected to different concentrations of UA for different periods. Early CKD rat model with elevated serum UA was established. Western blotting and quantitative real-time PCR (qPCR) were applied for measuring protein and mRNA expression of different cytokines. The animals were sacrificed and blood samples were collected for measurement of creatinine, UA, IL-1β, TNF-α, and ICAM-1. Renal tissues were pathologically examined by periodic acid-Schiff (PAS) or hematoxylin-eosin (HE) staining.ResultsThe expression of IL-1β, ICAM-1, NLRP3 complexes, and activation of NLRP3 inflammasome could be induced by UA, but the changes induced by UA were partially reversed by siRNA NLRP3 or caspase 1 inhibitor. Furthermore, we identified that UA regulated the activation of NLRP3 inflammasome by activating ROS and K+ efflux. In vivo results showed that UA caused the vascular endothelial injury by activating NLRP3/IL-1β pathway. While allopurinol could reduce UA level and may have protective effects on cardiovascular system.ConclusionsUA could regulate NLRP3/IL-1β signaling pathway through ROS activation and K+ efflux and further induce vascular endothelial cells injury in early stages of CKD.

Stem cell-based small-diameter vascular grafts in dynamic culture

ABSTRACT Purpose: Transplantation of autologous and/or allogeneic blood vessels is the most convenient treatment for vascular diseases. With regard to extensive need for blood vessels, developments in vascular tissue engineering are contributing greatly. In this study, our aim is to create intact small-diameter tubular vascular grafts cultivated in pulsatile flow bioreactor. Materials and Methods: CD146+ cell-based small-diameter vascular grafts were fabricated with ECM/glycosaminoglycans and polyurethane nanofibers. Characterization of the vascular graft was performed by SEM and WST-1. To mimic blood circulation in the bioreactor, human CD34+ cells cultured in megakaryocytes/platelets medium; then these cells were transferred inside of the vascular graft to mimic blood circulation. Cell differentiation was evaluated by flow cytometry and colony assay. Wright-Giemsa staining and polyploidy analysis were performed to show the differentiated cell population inside of the vascular graft. Anti-thrombogenic properties of the blood vessel were demonstrated by IF. Results: Polyurethane nanofibers provided a suitable environment for Human umbilical cord vein endothelial cells (HUVECs), and no significant cytotoxic effect was observed. Scanning electron microscopy (SEM) analysis of the tubular graft showed that under perfusion HUVECs, smooth muscle cells (SMCs) and fibroblasts formed layers that aligned on each other, respectively. The vascular graft was strong with a tensile strength of 0.70 MPa and elastic modulus of 0.007 GPa. When cultured in a bioreactor system, platelet adhesion to the vascular graft was remarkably low. Conclusion: In conclusion, this vascular graft may hold the potential to regenerate functional small-diameter vessels for cardiovascular tissue repair.

Tiotropium inhibits proinflammatory microparticle generation by human bronchial and endothelial cells

Tiotropium is a muscarinic antagonist that reduces the risk of acute exacerbations of chronic obstructive pulmonary disease, possibly through an as yet incompletely characterized anti-inflammatory activity. We hypothesized that muscarinic activation of bronchial epithelial cells and endothelial cells causes the release of proinflammatory microparticles and that tiotropium inhibits the phenomenon. Microparticle generation was assessed by a functional assay, by flow cytometry and by NanoSight technology. Immortalized bronchial epithelial cells (16HBE) and umbilical vein endothelial cells were treated with acetylcholine in the presence of varying concentrations of tiotropium. Intracellular calcium concentration, extracellular regulated kinase phosphorylation and chemokine content in the conditioned media were assessed by commercial kits. Acetylcholine causes microparticle generation that is completely inhibited by tiotropium (50 pM). Microparticles generated by acetylcholine-stimulated cells increase the synthesis of proinflammatory mediators in an autocrine fashion. Acetylcholine-induced upregulation of microparticle generation is inhibited by an inhibitor of extracellular regulated kinase phosphorylation and by a phospholipase C inhibitor. Tiotropium blocks both extracellular regulated kinase phosphorylation and calcium mobilization, consistent with the hypothesis that the drug prevents microparticle generation through inhibition of these critical pathways. These results might contribute to explain the effect of tiotropium in reducing acute exacerbations of chronic obstructive pulmonary disease.

Xanthoangelol Prevents Ox-LDL-Induced Endothelial Cell Injury by Activating Nrf2/ARE Signaling.

Atherosclerosis (AS) contributes to the development of several cardiovascular diseases such as myocardial infarction and stroke. Oxidized low-density lipoprotein (Ox-LDL)-induced endothelial cell injury plays a key role in the pathogenesis of AS. Thus, this study was conducted to examine the effects of a naturally occurring flavonoid compound, xanthoangelol (XAG), on Ox-LDL-induced cell injury. Human umbilical vein endothelial cells (HUVECs) were used as the in vitro cell model. The number of viable cells was determined using CCK-8 assay. Cell apoptosis was detected using Hoechst staining. Percentage of apoptotic cells was quantified by flow cytometry. The cellular levels of malondialdehyde (MDA), superoxide dismutase, catalase (CAT), and glutathione peroxidase were determined using enzyme-linked immunosorbent assays. The cellular reactive oxygen species level was detected by flow cytometry after fluorescence staining. The mRNA expression levels of nuclear factor-E2-related factor-2 (Nrf2), heme oxygenase-1 (HO-1), and NQO-1 were determined using quantitative real-time polymerase chain reaction assay. The protein levels of cleaved caspase-3, cleaved poly ADP-ribose polymerase, Bax, Bcl-2, Nrf2, Keap1, HO-1, and NQO-1 were measured by using Western blot assay. The HUVECs were transfected with Nrf2 siRNA to reduce the expression of Nrf2. XAG could effectively protect against Ox-LDL-stimulated cell death in HUVECs. These cytoprotective effects were due to its anti-apoptotic and anti-oxidant activities, as supported by the increase of SOD, CAT, and glutathione peroxidase activities, and the decrease of MDA and reactive oxygen species levels in injured HUVECs induced by Ox-LDL. Moreover, the results showed that XAG activated Nrf2/ARE signaling in a dose-dependent manner. Importantly, blockade of Nrf2 signaling using siRNA or specific inhibitor notably abolished the cytoprotective activities of XAG. These data suggest that XAG cytoprotects against Ox-LDL-induced cell injury through activating Nrf2/ARE-mediated antioxidative stress. Cumulatively, these findings show that EX has the potential to prevent and treat AS.

Mechanisms of Blood-Retinal Barrier Disruption by HIV-1.

It has been found that human immunodeficiency virus (HIV)-1 RNA or antigens can be detected in the intraocular tissues of HIV-1 patients even under effective highly active anti-retroviral therapy (HAART). In vivo, blood-retinal barrier (BRB) establishes a critical, physiological guardian against microbial invasion of the eye, but may be compromised in the presence of HIV-1. The envelope glycoprotein gp120 is exposed on the surface of the HIV envelope, essential for virus entry into cells by the attachment to specific cell surface receptors. The BRB disruption by glycoprotein gp120 has been widely recognized, which is toxic to human retinal epithelial cells (RPE) and umbilical vein endothelial cells (HUVEC). The present review elaborates on various mechanisms of BRB disruption induced by HIV gp120, which may represent potential targets for the prevention of ocular HIV complications in the future.

β-aminoisobutyric acid protects against vascular inflammation through PGC-1β-induced antioxidative properties

BackgroudAmong various myocyte-derived bioactive molecules (myokines), β-aminoisobutyric acid (BAIBA) is a unique myokine that attenuates skeletal muscle insulin resistance and inflammation, increases browning of white adipose tissue, and enhances hepatic fatty acid oxidation, resulting in upregulated energy expenditure of the whole body. In the present study, we investigated the effects of BAIBA on the vascular endothelial cell function. MethodsThe mRNA levels of proinflammatory molecules, antioxidants, and their related transcription regulators were examined by quantitative RT-PCR in BAIBA-treated human aortic or umbilical vein endothelial cells (HAEC or HUVEC, respectively), with or without tumor necrosis factor (TNF)-α stimulation. The protein expression and phosphorylation of AMP-activated protein kinase (AMPK) and endothelial nitric oxide synthase (eNOS) were determined by Western blot analysis. ResultsBAIBA pretreatment significantly suppressed the mRNA levels of the adhesion molecules in the TNF-α-stimulated HAEC and HUVEC. BAIBA treatment significantly increased the mRNA levels of antioxidant molecules, catalase, superoxide dismutases, thioredoxin, and gamma-glutamylcysteine ligases, together with mitochondrial biogenesis-related molecules, nuclear respiratory factor 1, and mitochondrial transcription factor A. In addition, BAIBA treatment significantly increased the transcription factors that regulated these genes [i.e., peroxisome proliferator-activated receptor (PPAR)-δ, PPAR-γ, estrogen-related receptor α (ERRα), and peroxisome proliferator-activated receptor gamma coactivator (PGC)-1β]. Adenovirus-mediated PGC-1β overexpression significantly increased the mRNA levels of all antioxidant molecules. The phosphorylation levels of AMPK and eNOS were unaltered by BAIBA. ConclusionsIn vascular endothelial cells, BAIBA had antiatherogenic effects through the PGC-1β−ERRα/PPAR-δ and PPAR-γ pathway. This can explain the beneficial effects of exercise on vascular endothelial function.

Role of mesenchymal stem cell-derived soluble factors and folic acid in wound healing.

Background/aimMesenchymal stem cells (MSCs) are a type of adult stem cell consisting of a heterogeneous subset of stromal stem cells that can be isolated from adult tissues. Folic acid is another important contributor to tissue regeneration and repair, which affects the synthesis of some building block molecules used for wound healing. In this study, we examine the effect of folic acid and MSC-derived soluble factors in the wound healing model.Materials and methodsHuman umbilical vein endothelial cells (HUVECs) and bone marrow-derived MSCs (BMSCs) were cultured for this study. Cell proliferation analysis was done with xCELLigence RTCA. After 48 h of cultivation, the cell culture medium was collected as MSC conditional medium containing mesenchymal stem cell-derived soluble factors (MDFs). Different concentrations of MDFs (12%, 25%, 50%, 75%, and 100%) were applied to the HUVEC cell line. Folic acid (25, 30, 50, 60, 75, 90, and 100 µM) was tested by application of three different groups (control, 25 µM folic acid, 625 µM folic acid inhibitors) for proliferation on the HUVEC cell line. The combined effects of folic acid and MDFs were tested on the HUVEC cell line with 25 µM folic acid and 50 µM MDFs. All data were statistically analyzed using SPSS 15.0 for Windows.ResultsSignificant differences were observed between controls and cells treated with folic acid, as well as between controls and both folic acid and MDFs (P < 0.05). Among the treated groups, the fastest wound closure rate was seen in cells treated with both folic acid and MDFs.ConclusionThe results show that both folic acid and MDFs increased the wound healing rate in HUVECs when they were used separately. The strongest benefits were seen in treatment using folic acid and MDFs together.

A Human Liver-on-a-Chip Platform for Modeling Nonalcoholic Fatty Liver Disease.

The liver possesses a unique microenvironment with a complex internal vascular system and cell-cell interactions. Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease, and although much effort has been dedicated to building models to target NAFLD, most in vitro systems rely on simple models failing to recapitulate complex liver functions. Here, an in vitro system is presented to study NAFLD (steatosis) by coculturing human hepatocellular carcinoma (HepG2) cells and umbilical vein endothelial cells (HUVECs) into spheroids. Analysis of colocalization of HepG2-HUVECs along with the level of steatosis reveals that the NAFLD pathogenesis could be better modeled when 20% of HUVECs are presented in HepG2 spheroids. Spheroids with fat supplements progressed to the steatosis stage on day 2, which could be maintained for more than a week without being harmful for cells. Transferring spheroids onto a chip system with an array of interconnected hexagonal microwells proves helpful for monitoring functionality through increased albumin secretions with HepG2-HUVEC interactions and elevated production of reactive oxygen species for steatotic spheroids. The reversibility of steatosis is demonstrated by simply stopping fat-based diet or by antisteatotic drug administration, the latter showing a faster return of intracellular lipid levels to the basal level.

The small GTPase Rab5c is a key regulator of trafficking of the CD93/Multimerin-2/\u03b21 integrin complex in endothelial cell adhesion and migration

BackgroundIn the endothelium, the single-pass membrane protein CD93, through its interaction with the extracellular matrix protein Multimerin-2, activates signaling pathways that are critical for vascular development and angiogenesis. Trafficking of adhesion molecules through endosomal compartments modulates their signaling output. However, the mechanistic basis coordinating CD93 recycling and its implications for endothelial cell (EC) function remain elusive.MethodsHuman umbilical vein ECs (HUVECs) and human dermal blood ECs (HDBEC) were used in this study. Fluorescence confocal microscopy was employed to follow CD93 retrieval, recycling, and protein colocalization in spreading cells. To better define CD93 trafficking, drug treatments and transfected chimeric wild type and mutant CD93 proteins were used. The scratch assay was used to evaluate cell migration. Gene silencing strategies, flow citometry, and quantification of migratory capability were used to determine the role of Rab5c during CD93 recycling to the cell surface.ResultsHere, we identify the recycling pathway of CD93 following EC adhesion and migration. We show that the cytoplasmic domain of CD93, by its interaction with Moesin and F-actin, is instrumental for CD93 retrieval in adhering and migrating cells and that aberrant endosomal trafficking of CD93 prevents its localization at the leading edge of migration. Moreover, the small GTPase Rab5c turns out to be a key component of the molecular machinery that is able to drive CD93 recycling to the EC surface. Finally, in the Rab5c endosomal compartment CD93 forms a complex with Multimerin-2 and active β1 integrin, which is recycled back to the basolaterally-polarized cell surface by clathrin-independent endocytosis.ConclusionsOur findings, focusing on the pro-angiogenic receptor CD93, unveil the mechanisms of its polarized trafficking during EC adhesion and migration, opening novel therapeutic opportunities for angiogenic diseases.

Melanocortin-1 Receptor Positively Regulates Human Artery Endothelial Cell Migration.

Melanocortin receptors (MCRs) belong to a hormonal signalling pathway with multiple homeostatic and protective actions. Microvascular and umbilical vein endothelial cells (ECs) express components of the melanocortin system, including the type 1 receptor (MC1R), playing a role in modulating inflammation and vascular tone. Since ECs exhibit a remarkable heterogeneity, we investigated whether human artery ECs express any functional MCR and whether its activation affects cell migration. We used reverse transcription real-time PCR to examine the expression of melanocortin system components in primary human artery ECs. We assessed MC1R protein expression and activity by western blot, immunohistochemistry, cAMP production, and intracellular Ca²⁺ mobilization assays. We performed gap closure and scratch tests to examine cell migration after stimulation with alpha-melanocyte-stimulating hormone (α-MSH), the receptor highest-affinity natural ligand. We assessed differential time-dependent transcriptional changes in migrating cells by microarray analysis. We showed that human aortic ECs (HAoECs) express a functionally active MC1R. Unlike microvascular ECs, arterial cells did not express the α-MSH precursor proopiomelanocortin, nor produced the hormone. MC1R engagement with a single pulse of α-MSH accelerated HAoEC migration both in the directional migration assay and in the scratch wound healing test. This was associated with an enhancement in Ca²⁺ signalling and inhibition of cAMP elevation. Time-course genome-wide expression analysis in HAoECs undergoing directional migration allowed identifying dynamic co-regulation of genes involved in extracellular matrix-receptor interaction, vesicle-mediated trafficking, and metal sensing - which have all well-established influences on EC motility -, without affecting the balance between pro- and anticoagulant genes. Our work broadens the knowledge on peripherally expressed MC1R. These results indicate that the receptor is constitutively expressed by arterial ECs and provide evidence of a novel homeostatic function for MC1R, whose activation may participate in preventing/healing endothelial dysfunction or denudation in macrovascular arteries.

Effect of lncRNA H19 on the apoptosis of vascular endothelial cells in arteriosclerosis obliterans via the NF-κB pathway.

To explore the effect of long non-coding ribonucleic acid (lncRNA) H19 on the apoptosis of vascular endothelial cells in arteriosclerosis obliterans (ASO) via the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. Human umbilical vein endothelial cells (HUVECs) were cultured, and lncRNA H19 was inhibited by Si-H9 and overexpressed by H19-OE. Then, the apoptosis rate was detected by flow cytometry, the target of lncRNA H19 was detected by dual luciferase reporter gene assay, and changes in the protein level were determined via Western blotting (WB). LncRNA H19 exhibited high expression in serum of patients with ASO, and compared with that in congeneric normal mice, the expression of lncRNA H19 in ASO mice rose. Besides, the proliferation ability of cells transfected with H19-OE was markedly strengthened, and H19-OE treatment could down-regulate the expression level of the apoptin, active cysteinyl aspartate-specific proteinase-3 (Caspase-3). In addition, lncRNA H19 bound to micro ribonucleic acid (miR)-19a in a targeted way. After lncRNA H19 was overexpressed, the expression of the NF-κB pathway key factors, p38 and p65, were notably increased, and the nuclear translocation of p65 was significantly enhanced after transfection with miR-19a. LncRNA H19 promotes the proliferation of vascular endothelial cells in ASO and inhibits the apoptosis of them via the NF-κB pathway.

Protein Tyrosine Phosphatase (PTP) 1B Inhibition Improves Endoplasmic Reticulum Stress‐Induced Apoptosis in Endothelial Cells

BackgroundEndoplasmic Reticulum (ER) stress response, which is activated in obesity and diabetes, contributes to the development of endothelial dysfunction in a complex interplay with insulin resistance. Previously, the inhibition of Protein Tyrosine Phosphatase 1B (PTP1B), a negative regulator of insulin receptor anchored to the ER membrane, was found to improved endothelial dysfunction; however, the role of PTP1B in ER stress‐mediated endothelial cell death and dysfunction is not fully understood.MethodsHuman Umbilical Vein Endothelial Cells (HUVECs) were exposed to Thapsigargin (100 nM; 5–24 h), a pharmacological ER stressor, in the presence and absence of PTP1B inhibitor (BML‐267; 20 μM) or PTP1B‐specific siRNA duplexes. Then, we have assessed the expression of ER stress markers [CCAAT‐enhancer‐binding protein homologous protein (CHOP) and Binding immunoglobulin protein (BiP)], angiogenic capacity of cells (Matrigel assay), cell cycle and apoptosis. Furthermore, we evaluated the expression of apoptotic effectors [cleaved caspases 3, 9, 12 and poly ADP ribose polymerase (PARP)‐1] and signaling pathways associated with cellular inflammation, cell survival and apoptosis: c‐Jun N‐terminal kinase (JNK), p42/p44 and p‐38 mitogen activated protein kinase (MAPK) in addition to autophagy (LC3‐II).ResultsOur findings highlighted a protective effect of PTP1B silencing on ER stress‐mediated endothelial cell apoptosis. Exposure of HUVECs with Thapsigargin caused as expected an increase in mRNA and protein expression of ER stress markers; however, the pharmacological inhibition of PTP1B partially prevented these effects. ER stress induction reduced the capacity of HUVECs to form tube‐like structures on a Matrigel matrix, a hallmark of endothelial dysfunction, while cells pre‐treated with PTP1B inhibitor were protected. The blockade of PTP1B using PTP1B inhibitor or specific siRNA, prevented ER stress‐induced apoptosis and cell cycle arrest at the G2/M phase. This focal effect of PTP1B inhibition on apoptosis was associated with reduced expression of cleaved caspases 9 and 12 and PARP‐1. The inhibition of PTP1B caused a concomitant increase in the phosphorylation of pro‐proliferative p42/p44 MPAK and a decrease in that of pro‐apoptotic p‐38 MAPK. Furthermore, the inhibition of PTP1B was associated with suppression of autophagy (low expression of LC3‐II) activated by Thapsigargin.ConclusionOur data bring further insight into the molecular mechanisms underpinning endothelial dysfunction in metabolic disorders such as obesity and diabetes. We specifically harnessed here the critical role of PTP1B, located at the surface of the ER, in ER stress‐mediated cell death and endothelial dysfunction through the activation of a complex signaling pathway involving multiple inflammatory responses including JNK, MPAK and autophagy.Support or Funding InformationThis work was funded by grants QUST‐1‐CPH‐2018‐2, QUST‐2‐CPH‐2017‐18 and QUUGCPH\2017‐1 from Qatar University to Dr. Abdelali Agouni.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tissue-Specific Influence of Lamin A Mutations on Notch Signaling and Osteogenic Phenotype of Primary Human Mesenchymal Cells.

Lamin A is involved in many cellular functions due to its ability to bind chromatin and transcription factors and affect their properties. Mutations of LMNA gene encoding lamin A affect the differentiation capacity of stem cells, but the mechanisms of this influence remain largely unclear. We and others have reported recently an interaction of lamin A with Notch pathway, which is among the main developmental regulators of cellular identity. The aim of this study was to explore the influence of LMNA mutations on the proosteogenic response of human cells of mesenchymal origin and to further explore the interaction of LMNA with Notch pathway. Mutations R527C and R471C in LMNA are associated with mandibuloacral dysplasia type A, a highly penetrant disease with a variety of abnormalities involving bone development. We used lentiviral constructs bearing mutations R527C and R471C and explored its influence on proosteogenic phenotype expression and Notch pathway activity in four types of human cells: umbilical vein endothelial cells (HUVEC), cardiac mesenchymal cells (HCMC), aortic smooth muscle cells (HASMC), and aortic valve interstitial cells (HAVIC). The proosteogenic response of the cells was induced by the addition of either LPS or specific effectors of osteogenic differentiation to the culture medium; phenotype was estimated by the expression of osteogenic markers by qPCR; activation of Notch was assessed by expression of Notch-related and Notch-responsive genes by qPCR and by activation of a luciferase CSL-reporter construct. Overall, we observed different reactivity of all four cell lineages to the stimulation with either LPS or osteogenic factors. R527C had a stronger influence on the proosteogenic phenotype. We observed the inhibiting action of LMNA R527C on osteogenic differentiation in HCMC in the presence of activated Notch signaling, while LMNA R527C caused the activation of osteogenic differentiation in HAVIC in the presence of activated Notch signaling. Our results suggest that the effect of a LMNA mutation is strongly dependent not only on a specific mutation itself, but also might be influenced by the intrinsic molecular context of a cell lineage.

Friend or Foe: A Cancer Suppressor MicroRNA-34 Potentially Plays an Adverse Role in Vascular Diseases by Regulating Cell Apoptosis and Extracellular Matrix Degradation.

BackgroundMicroRNAs (miRNAs) have emerged as central regulators of many processes. MiRNA-34 (miR-34) functions as a well-known tumor suppressor. The aim of this study was to investigate the mechanisms underlying how miR-34 participates in vascular disorders.Material/MethodsThree miR-34 family members (miR-34a, miR-34b, and miR-34c) were overexpressed or silenced in human vascular smooth muscle cells (VSMCs) and umbilical vein endothelial cells (UVECs), respectively, before the proliferation and apoptosis of cells were detected by using Cell Counting Kit-8 assay and Annexin V- fluorescein isothiocyanate/propidium iodide flow cytometry. The protein expression of apoptosis biomarkers was detected by western blot. Dual-luciferase reporter assay was performed to determine the candidate target of miR-34, and enzyme-linked immune sorbent assay was used to evaluate the effect of miR-34 on the expression of the target gene.ResultsOverexpression of miR-34 family members repressed proliferation and promoted apoptosis of VSMCs and UVECs, whereas miR-34 knockdown led to the opposite results. In addition, miR-34a inhibited the expression of alpha-1 antitrypsin (AAT), a serine protease inhibitor that suppresses the degradation of extracellular matrix, through a miR-34-binding site within the 3′-UTR of AAT.ConclusionsMiR-34 promoted apoptosis of VSMC and UVEC cells by inhibiting AAT expression. This finding provides an update on the understanding of the clinical value of miR-34, which might assist to uncover novel and effective therapeutic strategies for the treatment of vascular diseases.