Activity Of Human Umbilical Vein Endothelial Cells Research Articles

Picroside II, an iridoid glycoside from Picrorhiza kurroa, suppresses tumor migration, invasion, and angiogenesis in vitro and in vivo

Cancer is one of the leading causes of death worldwide. The development of novel anti-cancer agents from natural products is a promising approach to reduce cancer mortality. In this study, we investigated the anti-metastatic and anti-angiogenic activities of picroside II (PII) in human breast cancer cells both in vitro and in vivo. Our results demonstrated that PII significantly inhibited the migration and invasion of MDA-MB-231 cancer cells. With the treatment of PII, the activity of matrix metalloproteinase 9 (MMP-9) in MDA-MB-231 cancer cells was significantly inhibited both in vitro and in vivo. Meanwhile, PII showed effective anti-metastatic activity in an experimental lung metastasis model. Interestingly, cluster of differentiation 31 (CD31), a marker of angiogenesis, was significantly downregulated in the PII-treated tumor samples, indicating the anti-angiogenic activity of PII. Furthermore, we demonstrated that PII significantly inhibited the migration, invasion, and tube formation of human umbilical vein endothelial cells (HUVECs). The inhibition of MMP-9 activity in PII-treated HUVECs was also demonstrated. Finally, the suppression of angiogenesis by PII in the chick embryo chorioallantoic membrane (CAM) was observed. In conclusion, our results demonstrated that PII effectively inhibited the metastasis and angiogenesis of cancer cells both in vitro and in vivo, and thus, might be a novel candidate for cancer therapy.

Vanadium compounds induced damage of human umbilical vein endothelial cells and the protective effect of berberine.

This study was conducted to investigate the damage caused by vanadium compounds and to explore the protective effects of berberine (BBR) in human umbilical vein endothelial cells (HUVECs). BBR is a biologically active small molecule found in Coptis rhizome, a remedy used in traditional Chinese medicine to treat diabetes. BBR has also been shown to lower blood glucose in diabetic patients. MTT assay was performed to observe the influence of bis(acetylacetonato)-oxidovanadium [VO(acac)2] or sodium metavanadate (NaVO3) and BBR on viability of HUVECs. The monolayer permeability of the HUVECs was assessed by measuring the transendothelial electrical resistance (TER). The endothelial nitric oxide synthase (eNOS) activity was detected by ELISA. Flow cytometry was performed to detect the generation of reactive oxygen species (ROS). The results showed that the viability of HUVECs was decreased by treatment with vanadium compounds 50-400μM in a concentration-dependent manner, while 0.01-1μM BBR effectively protected HUVECs from the inhibitory effects of vanadium compounds on cell viability. Also 100 and 200μM VO(acac)2 induced high permeability and decreased eNOS activity in HUVECs. While 0.01-1μM BBR showed no improvement in the permeability, and failed to reverse the VO(acac)2-induced changes of eNOS activity, but BBR treatment increased the eNOS activity in control cells. The addition of 200μM VO(acac)2 significantly induced ROS generation in HUVECs, while 0.01 or 0.1 μM BBR reversed the change of ROS. In summary, BBR has protective effects in HUVECs damage induced by vanadium compounds, which is not mediated by eNOS, but related to reduced intracellular ROS.

Co-Culture of Osteoblasts and Endothelial Cells on a Microfiber Scaffold to Construct Bone-Like Tissue with Vascular Networks.

Bone is based on an elaborate system of mineralization and vascularization. In hard tissue engineering, diverse biomaterials compatible with osteogenesis and angiogenesis have been developed. In the present study, to examine the processes of osteogenesis and angiogenesis, osteoblast-like MG-63 cells were co-cultured with human umbilical vein endothelial cells (HUVECs) on a microfiber scaffold. The percentage of adherent cells on the scaffold was more than 60% compared to the culture plate, regardless of the cell type and culture conditions. Cell viability under both monoculture and co-culture conditions was constantly sustained. During the culture periods, the cells were spread along the fibers and extended pseudopodium-like structures on the microfibers three-dimensionally. Compared to the monoculture results, the alkaline phosphatase activity of the co-culture increased 3–6 fold, whereas the vascular endothelial cell growth factor secretion significantly decreased. Immunofluorescent staining of CD31 showed that HUVECs were well spread along the fibers and formed microcapillary-structures. These results suggest that the activation of HUVECs by co-culture with MG-63 could enhance osteoblastic differentiation in the microfiber scaffold, which mimics the microenvironment of the extracellular matrix. This approach can be effective for the construction of tissue-engineered bone with vascular networks.

Bach1-Induced Suppression of Angiogenesis Is Dependent on the BTB Domain

The transcription factor BTB and CNC homology 1 (Bach1) impairs angiogenesis after ischemic injury by suppressing Wnt/β-catenin signaling; however, the specific domains responsible for the antiangiogenic effects of Bach1 remain unclear. This study determined the role of the BTB domain of Bach1 in ischemic angiogenesis. Bach1 is highly expressed in circulating endothelial cells from acute myocardial infarction patients, and is the early induction gene after ischemic inuced injury. Mice were treated with adenoviruses coding for GFP (AdGFP), Bach1 (AdBach1), or a Bach1 mutant lacking the BTB domain (AdBach1-ΔBTB) after surgically induced hind-limb ischemia. Measures of blood-flow recovery, capillary density, and the expression of vascular endothelial growth factor (VEGF) were significantly lower in the AdBach1 group, but not in AdBach1-ΔBTB animals. Furthermore, transfection with AdBach1, but not AdBach1-ΔBTB, in human umbilical-vein endothelial cells (HUVECs) was associated with significant declines in 1) capillary density and hemoglobin content in the Matrigel-plug assay, 2) proliferation, migration, tube formation, and VEGF transcriptional activity and mRNA expression in HUVECs. Bach1 binds directly with TCF4, and this interaction is mediated by residues 81-89 of the Bach1 BTB domain and the N-terminal CTNNB1 domain of TCF4. Bach1, but not Bach1-ΔBTB, also copreciptated with histone deacetylase 1 (HDAC1), while the full-length HDAC1 proteins, but not HDAC1 mutants lacking the protein-interaction domain, co-precipitated with Bach1. Collectively, these results demonstrate that the anti-angiogenic activity of Bach1 is crucially dependent on molecular interactions that are mediated by the protein's BTB domain, and this domain could be a drug target for angiogenic therapy. Funding Statement: This work was supported by the Great Program (91639103 to D. Meng), and General Programs (81670450 and 81873469 to D. Meng, 81570081 and 81770083 to S. Li, 81800234 to L. Jiang, 81572713 to X. Zhi, 81873536 to X. Wang) of the National Natural Science Foundation of China, the National Key Research and Development Program of China (2018YFC1313600 to S. Li). Declaration of Interests: The authors state: None. Ethics Approval Statement: HUVECs were isolated (12) from the fresh umbilical cord of normal parturients with informed consent. The procedure was approved by the Ethics Committee of Experimental Research at Fudan University Shanghai Medical College. All experimental protocols were approved by the Ethics Committee of Experimental Research at Fudan University Shanghai Medical College and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication No 85-23).

Boiled Abalone Byproduct Peptide Exhibits Anti-Tumor Activity in HT1080 Cells and HUVECs by Suppressing the Metastasis and Angiogenesis in Vitro.

Abalone (Haliotis discus hannai) is a precious seafood in the market. It has been reported that biological active substances derived from abalone have anti-oxidative, anti-inflammatory, anti-bacterial, and anti-thrombosis potential. However, there were few studies to assess whether they have anti-cancer potential. In this study, we evaluated the anti-metastasis and anti-pro-angiogenic factors and mechanism of action of boiled abalone byproduct peptide (BABP, EMDEAQDPSEW) in human fibrosarcoma (HT1080) cells and human umbilical vein endothelial cells (HUVECs). The results demonstrated that BABP treatment significantly lowers migration and the invasion of HT1080 cells and HUVECs. BABP inhibits phorbol 12-myristate 13-acetate (PMA)-induced matrix metalloproteinase (MMP) expression and activity by blocking mitogen-activated protein kinases (MAPKs) and NF-κB signaling and hypoxia-induced vascular endothelial growth factor (VEGF) secretion and hypoxia inducible factor (HIF)-1α accumulation through suppressing the AKT/mTOR signal pathway. BABP treatment inhibits VEGF-induced VEGFR-2 expression and tube formation in HUVECs. The effect of BABP on anti-metastatic and anti-vascular activity in HT1080 cells and HUVECs revealed that BABP may be a potential pharmacophore for tumor therapy in the future.

Prokaryotic RNA activates endothelial cells promoting neutrophil transmigration.

Endothelial cell (EC)-neutrophil (PMN) interactions are crucial in the resolution of bacterial infections. Prokaryotic RNA (pRNA) has been reported as a pathogen-associated molecular pattern that is released from bacteria upon death and is able to activate PMN. In this work, we studied the effects of pRNA on EC and investigated whether these effects could modulate EC-PMN interaction. For this purpose, we purified total pRNA from Escherichia coli and used it as a stimulus for Human Umbilical Vein Endothelial Cells (HUVEC). We found that the incubation of pRNA with HUVEC caused the increase of surface intercellular adhesion molecule 1 (ICAM-1 or CD54) expression on HUVEC, and the secretion of IL-8 and von Willebrand factor, characteristics consistent with HUVEC activation, without causing toxic effects. Moreover, pRNA-treated HUVEC also induced PMN adhesion and the conditioned medium obtained from treated-HUVEC was chemotactic for PMN and caused their activation, as determined by CD11b upregulation. As reported previously, the degradation products of pRNA induced similar biological effects. The treatment of HUVEC with endocytosis inhibitors revealed that the entry of pRNA partially relied on a clathrin-dependent mechanism, whereas the effects of degradation products could not be inhibited by any of the inhibitors tested. Using a transwell system, we found that pRNA or degraded pRNA were also able to stimulate HUVEC when recognized from the basolateral side. Ourresults indicate that pRNA activates EC, resulting in the modulation ofEC-PMN interaction by inducing PMN chemotaxis, adhesion and activation. In the context of infection, pRNA sensed by EC and PMN could favor bacterial clearance.

RasGRP1 is a target for VEGF to induce angiogenesis and involved in the endothelial-protective effects of metformin under high glucose in HUVECs.

Impaired angiogenesis in endothelial cells is a hallmark of diabetes vascular complications. Ras guanine-releasing protein 1 (RasGRP1) is a guanine nucleotide exchange factor for Ras, and its role in endothelial angiogenesis has not been investigated. Given the importance of Ras in vascular endothelial growth factor (VEGF)-induced angiogenesis, we hypothesized that RasGRP1 may be a critical pathway downstream of VEGF and involved in endothelial angiogenesis. Furthermore, we investigate whether RasGRP1-dependent VEGF signaling was downregulated under high glucose conditions mimicking diabetes and required for the endothelial protective action of metformin in human umbilical vein endothelial cells (HUVECs). HUVECs were transfected with either RasGRP1 small interfering RNA (siRNA) or pEnter-RasGRP1 plasmid to down- and upregulate RasGRP1 expression before different treatments, such as added VEGF or not, exposed to high glucose (35 mM) or normal glucose (5 mM) in the presence or absence of metformin. Expression of VEGF, RasGRP1, and their signaling targets were analyzed by Western blot; migration and tube formation were detected by transwell chamber assay and Matrigel angiogenesis assay, respectively. Knockdown of RasGRP1 significantly attenuated VEGF-induced migration and tube formation activities of HUVECs and activation of AKT pathway. The expression of VEGF, RasGRP1, and AKT phosphorylation was downregulated in HUVECs exposed to high glucose compared with normal glucose, whereas metformin upregulated the RasGRP1-dependent VEGF signaling and ameliorates the impaired angiogenesis caused by high glucose. RasGRP1 is involved in the VEGF-induced angiogenesis and the pro-angiogenesis effects of metformin under hyperglycemia. © 2019 IUBMB Life, 71(9):1391-1400, 2019.

MiR-191 inhibit angiogenesis after acute ischemic stroke targeting VEZF1.

Acute ischemic stroke (AIS) is a major public health problem in China. Impaired angiogenesis plays crucial roles in the development of ischemic cerebral injury. Recent studies have identified that microRNAs (miRNAs) are important regulators of angiogenesis, but little is known the exact effects of angiogenesis-associated miRNAs in AIS. In the present study, we detected the expression levels of angiogenesis-associated miRNAs in AIS patients, middle cerebral artery occlusion (MCAO) rats, and oxygen-glucose deprivation/reoxygenation (OGD/R) human umbilical vein endothelial cells (HUVECs). MiR-191 was increased in the plasma of AIS patients, OGD/R HUVECs, and the plasma and brain of MCAO rats. Over-expression of miR-191 promoted apoptosis, but reduced the proliferation, migration, tube-forming and spheroid sprouting activity in HUVECs OGD/R model. Mechanically, vascular endothelial zinc finger 1 (VEZF1) was identified as the direct target of miR-191, and could be regulated by miR-191 at post-translational level. In vivo studies applying miR-191 antagomir demonstrated that inhibition of miR-191 reduced infarction volume in MCAO rats. In conclusion, our data reveal a novel role of miR-191 in promoting ischemic brain injury through inhibiting angiogenesis via targeting VEZF1. Therefore, miR-191 may serve as a biomarker or a therapeutic target for AIS.

Discovery of Dioxino[2,3-f]quinazoline derivative VEGFR-2 inhibitors exerting significant antipro-liferative activity in HUVECs and mice

Twelve 2,3-dihydro-[1,4]-dioxino[2,3-f]quinazoline derivatives were designed and evaluated as vascular endothelial growth factor receptor 2 (VEGFR-2) inhibitors. The most half-maximal inhibitory concentration (IC50) values of them were less than 10 nM. Among these compounds, 13d displayed highly effective inhibitory activity against VEGFR-2 (IC50 = 2.4 nM) and excellent antiproliferative activities against human umbilical vein endothelial cells (HUVECs) (IC50 = 1.2 nM). When anti-tumor animal experiments were carried out in mice, the tumor almost disappeared (TGI = 133.0%) after six days of administration of 13d. Therefore, 13d was a potential and effective anticancer agent. The binding conformations were respectively compared between VEGFR-2 with 13d and leading compound lenvatinib, and shows that they have similar binding modes.

Astragaloside IV exerts angiogenesis and cardioprotection after myocardial infarction via regulating PTEN/PI3K/Akt signaling pathway

Aims and methodsAcute myocardial infarction (AMI) is a common cardiovascular disease with high mortality. Astragaloside IV (AS-IV) was reported to have cardioprotective effect after AMI. We hypothesize that the cardioprotective role of AS-IV is exerted by enhancing angiogenesis via regulating PTEN/PI3K/Akt signaling pathway. To valid our hypothesis, AMI rats and human umbilical vein endothelial cells (HUVECs) were employed in our study. Key findingsAfter treatment, cardiac function, survival rate, infarct size, pathological changes and fibrosis, cell apoptosis, ultrastructural changes, angiogenesis and expression of PTEN/PI3K/Akt signaling pathway were evaluated, respectively. In vitro study we detected proliferation, tube formation and signaling pathway activation of HUVECs treated with AS-IV, lentivirus overexpressed PTEN was employed to elucidate the potential mechanism. The results indicated that AS-IV administration significantly improved cardiac function and survival rate, limited infarct size, ameliorated pathological changes and fibrosis deposition, inhibited apoptosis, relieved ultrastructure injury and enhanced angiogenesis, PTEN/PI3K/Akt signaling pathway was activated simultaneously compared to the model group. In vitro study suggested that AS-IV treatment promoted cell proliferation and tube formation, and induced PTEN/PI3K/Akt signaling pathway activation. Importantly, overexpression of PTEN by lentivirus abolished AS-IV-induced angiogenesis. SignificanceOur study indicated that AS-IV could promote angiogenesis and cardioprotection after myocardial infarction. The mechanisms involve activation of PTEN/PI3K/Akt signaling pathway.

Curcumin inhibits angiogenesis in endothelial cells using downregulation of the PI3K/Akt signaling pathway

Curcumin, a major yellow pigment and active component of turmeric, has anti-angiogenesis properties. However, its molecular targets and mechanisms of curcumin effects on angiogenesis of endothelial cells are not known. The effect of curcumin on angiogenesis activity of endothelial cells in vitro was studied. Human umbilical vein endothelial cells (HUVEC) were mixed with various concentrations of curcumin for 72 h. Cell viability was measured using the WST-1 test. The in vitro tubulogenesis assay was used to determine the potency of curcumin in inhibiting capillary-like formation. HUVEC migration capacity was evaluated using the scratch and transwell insert assays. Lipoprotein lipase activity of curcumin-treated HUVEC was measured. Vascular endothelial growth factor-1 (VEGFR-1) and −2 levels, and the phospo-endothelial nitric oxide synthase (p-eNOS)/eNOS ratio were measured using an ELISA test. The metalloproteinase activity was evaluated using gelatin zymography. The ratio of p-Akt/Akt was followed using western blotting. The WST-1 assay indicated a suppressed proliferation of HUVEC after exposure to curcumin in a dose- and time-dependent manner. Curcumin was able to diminish tube area and length values in HUVEC on Matrigel substrate. A significant reduction in migration rate of curcumin-treated cells coincided with diminished MMP-2 and MMP-9 activities. Lipoprotein lipase activity of HUVEC was reduced following treatment with curcumin indicated by a diminished fluorescence activity. The ELISA test showed a down-regulation of VEGFR-2. Western blotting confirmed the reduced p-Akt/Akt ratio after HUVEC being exposed to curcumin. Curcumin is able to inhibit angiogenesis through the modulation of VEGFR and PI3K/Akt signaling pathway.

Astragaloside IV Protects Against Oxidized Low-Density Lipoprotein (ox-LDL)-Induced Endothelial Cell Injury by Reducing Oxidative Stress and Inflammation.

BackgroundEndothelial injury is the main mechanism of atherosclerosis, and is caused by oxidized low-density lipoprotein (ox-LDL). Astragaloside IV (AS-IV) is the primary active ingredient of the Chinese herb Huangqi, and exhibits antioxidant and anti-inflammatory properties in cardiovascular diseases. This study investigated the protective effect of AS-IV in human umbilical vein endothelial cells (HUVECs).Material/MethodsHUVEC cells were induced with ox-LDL to establish an in vitro atherosclerosis model. Then HUVECs were pretreated for 1 h with AS-IV at different concentrations (10, 20, and 50 μM) and then exposed to ox-LDL (100 μg/mL) for 48 h. The cell viability, lactate dehydrogenase (LDH) release, apoptosis, migration, intracellular reactive oxygen species (ROS), and NADPH oxidase activity of HUVECs were measured. qRT-PCR was performed to measure the mRNA expressions of Nrf2, HO-1, TNFα, and IL-6. Enzyme-linked immunosorbent assay (ELISA) was performed to measure the supernatant contents of TNFα and IL-6.ResultsExposure of HUVECs to ox-LDL reduced cell viability and migration, induced apoptosis, and increased intracellular ROS production and NADPH oxidase. Pretreatment with AS-IV (10, 20, and 50 μM) significantly enhanced the cell viability and migration, suppressed LDH release, apoptosis, ROS production, and NADPH oxidase in HUVECs, in a concentration-dependent manner. The AS-IV (50 μM) alone did not show significant differences from control. AS-IV increased mRNA expressions of Nrf2 and HO-1 and decreased mRNA expressions of TNFα and IL-6 in the ox-LDL-HUEVC cells. Furthermore, AS-IV reduced supernatant contents of TNFα and IL-6.ConclusionsAstragaloside IV prevents ox-LDL-induced endothelial cell injury by reducing apoptosis, oxidative stress, and inflammatory response.

Impaired Bone Regenerative Effect of Exosomes Derived from Bone Marrow Mesenchymal Stem Cells in Type 1 Diabetes.

Stem cell‐derived exosomes have exhibited promise for applications in tissue regeneration. However, one major problem for stem cell‐derived exosome therapies is identifying appropriate source cells. In the present study, we aimed to compare the bone regenerative effect of exosomes secreted by bone marrow mesenchymal stem cells (BMSCs) derived from type 1 diabetes rats (dBMSC‐exos) and exosomes secreted by BMSCs derived from normal rats (nBMSC‐exos). BMSCs were isolated from rats with streptozotocin‐induced diabetes and normal rats. dBMSC‐exos and nBMSC‐exos were isolated by an ultracentrifugation method and identified. The effects of dBMSC‐exos and nBMSC‐exos on the proliferation and migration of BMSCs and human umbilical vein endothelial cells (HUVECs) were investigated. The effects of exosomes on the osteogenic differentiation of BMSCs and the angiogenic activity of HUVECs were compared. Finally, a rat calvarial defect model was used to compare the effects of exosomes on bone regeneration and neovascularization in vivo. In vitro, dBMSC‐exos and nBMSC‐exos both enhanced the osteogenic differentiation of BMSCs and promoted the angiogenic activity of HUVECs, but nBMSC‐exos had a greater effect than dBMSC‐exos. Similarly, in vivo, both dBMSC‐exos and nBMSC‐exos promoted bone regeneration and neovascularization in rat calvarial defects, but the therapeutic effect of nBMSC‐exos was superior to that of dBMSC‐exos. The present study demonstrates for the first time that the bone regenerative effect of exosomes derived from BMSCs is impaired in type 1 diabetes, indicating that for patients with type 1 diabetes, the autologous transplantation of BMSC‐exos to promote bone regeneration may be inappropriate. stem cells translational medicine 2019;8:593–605

Exposure to tris(1,3-dichloro-2-propyl) phosphate (TDCPP) induces vascular toxicity through Nrf2-VEGF pathway in zebrafish and human umbilical vein endothelial cells

The growing production and extensive use of organophosphate flame retardants (OPFRs) have led to an increase in their environmental distribution and human exposure. Developmental toxicity is a major concern of OPFRs' adverse health effects. However, the impact of OPFRs exposure on vascular development and the toxicity pathway for developmental defects are poorly understood. In this study, we investigated the effects of exposure to tris(1,3-dichloro-2-propyl) phosphate (TDCPP), a frequently detected OPFR, on early vascular development, and the possible role of nuclear factor erythroid 2-related factor (Nrf2)-dependent angiogenic pathway in TDCPP's vascular toxicity. TDCPP exposure at 300 and 500 μg/L impeded the growth of intersegmental vessels (ISV), a type of microvessels, as early as 30 hpf. Consistently, a similar pattern of decreased extension and remodeling of common cardinal vein (CCV), a typical macrovessel, was observed in zebrafish at 48 hpf and 72 hpf. Developing vasculature in zebrafish was more sensitive than general developmental parameters to TDCPP exposure. The expression of genes related to VEGF signaling pathway dose-dependently decreased in TDCPP-treated larvae. In in vitro experiments using human umbilical vein endothelial cells (HUVECs), the increased cell proliferation induced by VEGF was suppressed by TDCPP exposure in a dose-dependent fashion. In addition, we found a repression of Nrf2 expression and activity in TDCPP-treated larvae and HUVECs. Strikingly, the application of CDDO-Im, a potent Nrf2 activator, enhanced VEGF and protected against defective vascular development in zebrafish. Our results reveal that vascular impairment is a sensitive index for early exposure to TDCPP, which could be considered in the environmental risk assessment of OPFRs. The identification of Nrf2-mediating VEGF pathway provides new insight into the adverse outcome pathway (AOP) of OPFRs.

Propofol activates autophagic activity of vascular endothelial cells by inhibiting SENP1 expression and attenuates vascular endothelial injury induced by ischemia-reperfusion in orthopedic surgery

The aim of this study was to examine the effect and mechanism of SENP1 in the protection of vascular endothelial cells by propofol during ischemia-reperfusion (IR) injury. Thirty-eight patients undergoing minor lower limb orthopedic surgery under general anesthesia were included, and divided evenly into sevoflurane group and propofol group. Peripheral blood was collected before fixing tourniquets (T0 time point) and one hour after releasing tourniquets (T1 time point). Contents of endothelin, vWF and malondialdehyde were determined by enzyme-linked immunosorbent assay to evaluate vascular injury. Quantitative real-time polymerase chain reaction was used to determine mRNA expression; western blotting, to determine protein expression; CCK-8 assay, Transwell assay and flow cytometry, to examine the proliferation, migration and apoptosis of human umbilical vein endothelial cells (HUVECs); western blotting and laser scanning confocal microscopy, to analyze autophagy of HUVECs; protein immunoprecipitation, to examine the relationship between SENP1 and the SUMOylation of autophagy-associated VPS34 protein. The results showed that the contents of peripheral blood vessel injury markers and the expression of SENP1 were elevated when vascular injury was induced by IR in patients undergoing orthopedic surgery, whereas propofol reduced these levels. Inhibition of SENP1 expression reduced injury of HUVECs induced by IR. Silencing expression of SENP1 promoted autophagy of HUVECs. SENP1 promoted deSUMOylation of VPS34, leading to reduced autophagy of HUVECs. The study demonstrates that propofol activates autophagic activity of HUVECs by inhibiting SENP1 expression and attenuates vascular endothelial injury induced by IR in orthopedic surgery. This effect may be related to SENP1 regulating the deSUMOylation of VPS34.

Exosomes Derived From Low‐Intensity Pulsed Ultrasound‐Treated Dendritic Cells Suppress Tumor Necrosis Factor–Induced Endothelial Inflammation

Endothelial cell inflammation plays an important role in atherosclerosis. Low-intensity pulsed ultrasonography (LIPUS) exerts an anti-inflammatory function on endothelial cells, whereas the underlying mechanism has not been fully elucidated. Bone marrow dendritic cells (BMDCs) derived from bone barrow cells were treated with LIPUS, and exosomes secreted into the supernatant were purified. The isolated exosomes were incubated with human umbilical vein endothelial cells (HUVECs) to investigate their effect on tumor necrosis factor (TNF)-α-induced endothelial inflammation. Ultrastructure was analyzed by transmission electron microscopy. Messenger RNA levels were determined by quantitative reverse transcription polymerase chain reaction, and protein levels were analyzed by western blot. The isolated exosomes presented a typical exosomal size of 30 to 100 nm in diameter and expressed exosome positive markers (Alix, CD63, and TSG101) but not the exosome negative marker (Calnexin). Exosomes derived from LIPUS-treated BMDCs were rich in miR-16 and miR-21, which could be engulfed by HUVECs. Pretreatment with exosomes impeded TNFα-induced HUVEC activation and downregulated TNFα-stimulated expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, thus preventing TNFα-induced activation of the nuclear factor-κB signaling pathway. Exosomes derived from LIPUS-treated BMDC inhibit TNFα-induced endothelial inflammation by inhibiting the nuclear factor-κB signaling pathway.

Lixisenatide enhances mitochondrial biogenesis and function through regulating the CREB/PGC-1α pathway

Mitochondrial dysregulation has been associated with vascular endothelial dysfunction and pathophysiological development of cardiovascular diseases. Lixisenatide is a drug approved by the US Food and Drug Administration for the treatment of type 2 diabetes (T2D). Little information regarding the effects of lixisenatide on mitochondrial function in endothelial cells has been reported before. In the current study, we found that treatment with lixisenatide significantly increased the expression of PGC-1α, a “molecular switch” of mitochondrial biogenesis in human umbilical vein endothelial cells (HUVECs). Lixisenatide treatment also promoted the expressions of NRF1 and TFAM, which are the target genes of PGC-1α and executors of mitochondrial biogenesis. Importantly, our results indicate that lixisenatide treatment promoted mitochondrial biogenesis by elevating the ratio of mitochondrial-to-nuclear DNA (mtDNA/nDNA), mitochondrial mass, cytochrome B expression, and citrate synthase activity in HUVECs. Correspondingly, we found that lixisenatide treatment led to a functional gain and improvement in mitochondria by increasing the mitochondrial respiration rate and ATP generation. Mechanistically, lixisenatide treatment induced the phosphorylation of CREB at Ser133. Blockage of CREB phosphorylation using its inhibitor H89 abolished the effects of lixisenatide on the activation of PGC-1α/NRF-1/TFAM as well as the increase in mtDNA/nDNA. These findings suggest that lixisenatide promoted mitochondrial biogenesis in endothelial cells through activating the PGC-1α signaling pathway, which is mediated by the transcriptional factor CREB.

Paeoniflorin attenuates oxidized low-density lipoprotein-induced apoptosis and adhesion molecule expression by autophagy enhancement in human umbilical vein endothelial cells.

Oxidized low-density lipoprotein (ox-LDL)-induced endothelial dysfunction is recognized as a driving force in the development of atherosclerosis (AS). Paeoniflorin (Pae), a typical traditional herbal medicine, possesses anti-inflammatory, antioxidative, antihyperglycaemic, and antiapoptotic properties. Our study aimed to investigate the effects of Pae on ox-LDL-induced injury of the human umbilical vein endothelial cells (HUVECs) and to explore its molecular mechanism. We found that ox-LDL stimulation inhibited cell viability, activated autophagy, and induced apoptosis and adhesion molecule expression in HUVECs. Pae rescued ox-LDL-induced viability reduction and enhanced the ox-LDL-induced autophagy activation in HUVECs. Pae inhibited ox-LDL-induced apoptosis and adhesion molecule expression by autophagy enhancement in HUVECs. In addition, inhibition of SIRT1 by EX-527 abolished the promoting effect of Pae on autophagy and restored the inhibitory effect of Pae on apoptosis and adhesion molecule expression in the presence of ox-LDL. In conclusion, Pae attenuated ox-LDL-induced apoptosis and adhesion molecule expression by autophagy enhancement via upregulation of SIRT1 in HUVECs, shedding light on the mechanism underlying the protective effect of Pae on ox-LDL-induced injury of HUVECs.

Activation of Keap1-Nrf2 signaling by 4-octyl itaconate protects human umbilical vein endothelial cells from high glucose

High glucose (HG) induces oxidative injury to cultured human umbilical vein endothelial cells (HUVECs). Recent studies have discovered 4-octyl itaconate (OI) as a novel and cell permeable Nrf2 (nuclear-factor-E2-related factor 2) activator. Its potential activity in HG-treated HUVECs was tested here. In HUVECs OI disrupted Keap1-Nrf2 association, causing Nrf2 protein accumulation and nuclear translocation, as well as transcription and expression of Nrf2-ARE-dependent genes, including HO1, NQO1 and GCLM. Significantly, pretreatment with OI potently inhibited HG (40 mM glucose)-induced death and apoptosis of HUVECs. Moreover, OI potently inhibited HG-induced reactive oxygen species (ROS) production, lipid peroxidation, superoxide accumulation and mitochondrial depolarization in HUVECs. Activation of Nrf2 is required for OI-induced cytoprotection in HUVECs. Nrf2 shRNA or knockout (by CRISPR/Cas9 method) reversed OI-mediated HUVEC protection against HG. Further studies showed that Keap1 silencing or Cys151S mutation mimicked and nullified OI-induced activity in HUVECs. Taken together, we conclude that OI activates Keap1-Nrf2 signaling to protect HUVECs from HG.

Protective Effects of Carbon Monoxide Delivered By Corm-401 in Hyperhemolysis in Patients with Sickle Cell Disease

IntroductionHyperhemolysis has been well described in delayed hemolytic transfusion reaction (DHTR), which is one of the most serious complications of transfusion especially in patients with sickle cell disease (SCD). In the most severe cases, this life-threatening syndrome may reach 10% lysis of total red blood cells (RBCs) (Habibi A et. al. Am J Hematol 2016). DHTR is characterized by an acute anemia with increased plasma free Hb and free heme. Hyperhemolysis can also be found in other hemoglobinopathies and extracorporeal circulation procedures.Most in vitro studies on hyperhemolysis used only purified Hb or heme which did not allow to investigate all deleterious effects on the endothelium. We have developed a global and physiopathologic approach to assess the mechanism of hyperhemolysis-induced endothelial dysfunction. For this purpose, we created a microfluidic model reproducing hyperhemolysis observed in the most severe DHTR but it's also applicable for other pathogenesis of hyperhemolysis.Among the potential therapeutic approaches, carbon monoxide (CO) is beneficial on endothelial cells by its anti-inflammatory, antioxidant and vasodilator effect which reduce the toxicity of Hb and improve tissue perfusion (Motterlini R et.al. Nat. Rev. Drug Discov 2010). Here we investigated the therapeutic effects of a CO-releasing molecule, CORM-401, in hyperhemolysis-induced endothelial dysfunction.Materials and methodsFlow culture Human Umbilical Vein Endothelial Cells (HUVECs) in fibronectin-coated µ-Slides were preconditioned for 4 hours under shear stress of 1dyn/cm² with sonicated RBCs of healthy donors (AA) reconstituted in serum of either AA or SCD patients (SS) at 7 g/L of free Hb. In negative control conditions, cells were pretreated similarly with either AA serum or culture medium. Cells were then collected for analyses of actin network, membrane markers and endothelial function. Treatment with CORM-401 or inactive CORM (iCORM) was performed at fixed concentration during the preconditioning and perfusion steps.ResultsHemolysis induces multiple endothelial damage and dysfunctionAfter exposure to hemolysate, we noted a significant increase of subendothelial matrix exposition (fibronectin-coated surfaces) due to actin network reorganization and cells detachment. Immunofluorescence staining showed a moderate activation of HUVECs induced by hemolysate with membrane expression of adhesion molecules such as ICAM-1, E-Selectin and VCAM-1, but not VWF and P-Selectin. At a similar free Hb level, preconditioning with hemolysate without microparticles (MPs) had the lowest deleterious impact on HUVECs suggesting a role of MPs in hyperhemolysis.Perfusion of AA whole blood on HUVECs pretreated with hemolysate resulted in aggregation and activation of platelets in a GPIIbIIIa-dependent manner at injury sites. We also observed a significant increase of adhered RBCs (14-fold compared to control).Compared to whole blood RBC lysis, purified Hb induced similar subendothelial exposure and RBC adherence but did not lead to significant HUVECs activation.To study the DHTR in RBC transfusion of SCD patients, SS serum was used to perform hemolysis preconditioning of HUVECs. Adherence of RBCs was similar to condition using AA serum suggesting that endothelial damage in DHTR of SCD patients depend on hemolysate compositions and RBC MPs, rather than on patients' serum.Effects of CORM-401 on hemolysis-induced endothelial dysfunctionAdding CORM-401 (50 to 100 µM) to the hemolysate produced 15% free COHb leading to a concentration-dependent decrease in hemolysis-induced HUVECs activation and RBC adherence (Figure 1 C, E). While no significant effects at the subendothelial exposure were observed, the aggregation of platelets at injury sites was decreased after CORM-401 treatment compared to controls (Figure 1 A, B, D), confirming the anti-platelet aggregation effect of this molecule (Chlopicki S et. al. Naunyn Schmiedebergs Arch Pharmacol 2012).ConclusionOur in vitro model enabled the reproduction of endothelial damage by hyperhemolysis and to determine the deleterious effects of each hemolysate component. The in vitro beneficial effects of CORM-401 were also demonstrated and an in vivo study on SCD mice is underway to explore the therapeutic effects of this molecule against hyperhemolysis-induced endothelial damage. [Display omitted] DisclosuresBartolucci:Addmedica: Research Funding; GBT: Membership on an entity's Board of Directors or advisory committees; Fondation Fabre: Research Funding; Novartis US: Membership on an entity's Board of Directors or advisory committees.