Inhibited Human Umbilical Vein Endothelial Cells Proliferation Research Articles

Activating autophagy and ferroptosis of 3‑Chloropropane‑1,2‑diol induces injury of human umbilical vein endothelial cells via AMPK/mTOR/ULK1.

3‑Chloropropane‑1,2‑diol (3‑MCPD) is an internationally recognized food pollutant. 3‑MCPD has reproductive, renal and neurotoxic properties. However, whether 3‑MCPD induces human umbilical vein endothelial cell (HUVEC) injury has not been previously reported. In the present study, HUVECs were treated using 2µg/ml 3‑MCPD for 24h at 37˚C. The effects of 3‑MCPD on HUVEC proliferation and cell cycle arrest, death and senescence were then assessed using Cell Counting Kit‑8 (CCK‑8), flow cytometry and β‑galactosidase staining, respectively. Whether 3‑MCPD induced ferroptosis was evaluated using JC‑1 and FerroOrange staining and transmission electron microscopy. A small interfering RNA targeting AMPK was used to assess whether 3‑MCPD promoted ferroptosis via AMPK signaling. The results demonstrated that 3‑MCPD inhibited HUVEC proliferation in a dose‑dependent manner and induced cell cycle arrest. Furthermore, 3‑MCPD promoted senescence in HUVECs with elevated DNA damage and cell death. The CCK‑8 results demonstrated that ferroptosis and autophagy inhibitors significantly reversed cell death caused by 3‑MCPD. Moreover, 3‑MCPD increased mitochondrial membrane potential, which indicated that 3‑MCPD contributed to mitochondrial dysfunction. 3‑MCPD also markedly increased intracellular Fe2+ levels and lipid peroxidation in HUVECs. The present study assessed the underlying mechanism by which 3‑MCPD activated autophagy and ferroptosis in HUVECs. The data demonstrated that 3‑MCPD significantly increased phosphorylation levels of AMPK and unc‑51 like autophagy activating kinase (ULK1) but significantly decreased phosphorylation of mTOR in HUVECs. Furthermore, silencing of AMPK significantly reversed the increase in autophagy, lipid peroxidation and Fe2+ induced by 3‑MCPD. In conclusion, 3‑MCPD demonstrated a significant damaging effect on HUVECs via induction of autophagy and ferroptosis; such effects may be mediated by AMPK/mTOR/ULK1 signaling. To the best of our knowledge, the present study was the first to demonstrate the mechanism of 3‑MCPD‑induced vascular endothelial cell injury and lays a molecular foundation for the prevention of 3‑MCPD‑related vascular diseases.

Influence of placental exosomes from early onset preeclampsia women umbilical cord plasma on human umbilical vein endothelial cells.

Early onset preeclampsia (EOSP, PE) is characterized by hypertension, proteinuria, and endothelial dysfunction. Oxidative stress-induced trophoblast dysfunction is a major pathology in PE. Placental exosomes are extracellular vesicles that are involved in "mother-placenta-foetal communication" and can regulate the biological functions of endothelial cells. Our study was designed to evaluate placental exosomes effects on endothelial cells. Umbilical cord blood from normal pregnant women and patients with PE were collected. A hypoxia/reoxygenation (H/R) model in human first trimester extravillous trophoblast cell (HTR8/SVneo) line to simulate the PE model of oxidative stress in vitro. Then, placental exosomes (i.e., NO-exo, H/R-exo, N-exo, and PE-exo) were extracted and identified. Finally, the effects of placental exosomes on the biological functions of human umbilical vein endothelial cells (HUVECs) were further evaluated by performing a series of experiments. Placental exosomes had a double-membrane cup structure with diameters of 30-150 nm, and there was no obvious difference in placental exosomes. Compared with NO-exo and N-exo, H/R-exo and PE-exo inhibited HUVECs proliferation, tube formation and migration, increased permeability and apoptosis in vitro. We hypothesize that H/R-exo and PE-exo impair vessel development by disrupted biological functions in endothelial cells, which may result in vascular disorders in offspring.

TRPV3 promotes the angiogenesis through HIF-1α-VEGF signaling pathway in A549 cells

BackgroundAngiogenesis is an essential physiological process in the growth and metastasis of primary tumors. Ca2+ signaling is crucial for tumor angiogenesis. The purpose of this study was to detect the potential role of Ca2+ permeable transient receptor potential vanilloid-3 (TRPV3) in the angiogenesis of non-small cell lung cancer (NSCLC). MethodsSmall interfering RNA was used to down-regulate TRPV3 expression in A549 cells. A laser scanning confocal microscope was used to examine intracellular calcium concentration ([Ca2+]i). Human umbilical vein endothelial cells (HUVECs) tube formation and migration assay, Western blot, MTT and ELISA were performed to detect the potential mechanisms of TRPV3 in tumor angiogenesis. A mouse tumor xenograft model was performed to expound the effects of TRPV3 on tumor cell growth. ResultsInhibition of TRPV3 reduced [Ca2+]i and protein expressions of VEGF and HIF-1α in A549 cells. Moreover, HIF-1α depletion decreased the secretion and expression of VEGF. Depletion of TRPV3 inhibited HUVECs proliferation, tube formation and migration induced by conditioned medium. And TRPV3 inhibition could decrease the volume of xenograft tumors and MVD of CD34+ cells. The expression levels of HIF-1α, VEGF and p-CaMKП in the xenograft tumors in RuR and siTRPV3 groups was reduced. ConclusionsTRPV3 calcium channel protein may play a key role in NSCLC angiogenesis. TRPV3 could promote the angiogenesis through HIF-1α-VEGF signaling pathway. Targeting TRPV3 channel protein by novel approaches would be useful for reversing NSCLC angiogenesis.

Human placenta mesenchymal stem cell-derived exosome shuttling microRNA-130b-3p from gestational diabetes mellitus patients targets ICAM-1 and perturbs human umbilical vein endothelial cell angiogenesis.

The aim of this study was to investigate the roles of miR-130b-3p and ICAM-1 in gestational diabetes mellitus (GDM) and their potential association. Human placenta mesenchymal stem cells (PlaMSCs) were isolated from GDM patients, and the effects of the PlaMSCs from GDM patients (GDM-MSCs) and the exosomes secreted by GDM-MSCs on human umbilical vein endothelial cell (HUVEC) proliferation, migration, and angiogenesis were detected. Next, GDM-MSCs were transfected with miR-130b-3p antagomir to modify miR-130b-3p expression in GDM-MSCs-derived exosomes, and the exosomes with modified miR-130b-3p expression were cultured with HUVECs to evaluate exosomal miR-130b-3p on HUVEC function. Furthermore, a target gene of miR-130b-3p was predicted and assessed. The miR-130b-3p-modified exosomes were cultured with HUVECs transfected with ICAM-1 shRNA to determine the effect of miR-130b-3p-ICAM-1 crosstalk on HUVEC function. Additionally, a GDM mouse model was conducted to further study the effect of miR-130b-3p in GDM in vivo. GDM-MSCs inhibited HUVEC proliferation and angiogenesis. The elevated expression of miR-130b-3p was found in GDM-MSCs-derived exosomes. GDM-MSCs-derived exosomes repressed the proliferation and angiogenesis of HUVECs and miR-130b-3p inhibition could restrain the inhibition of the exosomes on HUVEC function. Mechanistically, miR-130b-3p downregulated ICAM-1 expression in a targeted manner, and thereby enhanced HUVEC proliferation, migration, and angiogenesis and increased the expression of angiogenesis-related factors. Moreover, miR-130b-3p inhibition promoted placental angiogenesis in GDM mice and upregulated ICAM-1 expression. Conclusively, GDM-MSCs-derived exosomes shuttling miR-130b-3p repressed proliferation, migration, and angiogenesis of HUVECs by regulating ICAM-1 expression.

Pharmacological Approaches to Attenuate Inflammation and Obesity with Natural Products Formulations by Regulating the Associated Promoting Molecular Signaling Pathways.

Obesity is a public health problem characterized by increased body weight due to abnormal adipose tissue expansion. Bioactive compound consumption from the diet or intake of dietary supplements is one of the possible ways to control obesity. Natural products with adipogenesis-regulating potential act as obesity treatments. We evaluated the synergistic antiangiogenesis, antiadipogenic and antilipogenic efficacy of standardized rebaudioside A, sativoside, and theasaponin E1 formulations (RASE1) in vitro in human umbilical vein endothelial cells (HUVECs), 3T3-L1 preadipocytes respectively, and in vivo using a high-fat and carbohydrate diet-induced obesity mouse model. Orlistat was used as a positive control, while untreated cells and animals were normal controls (NCs). Adipose tissue, liver, and blood were analyzed after dissection. Extracted stevia compounds and green tea seed saponin E1 exhibited pronounced antiobesity effects when combined. RASE1 inhibited HUVEC proliferation and tube formation by suppressing VEGFR2, NF-κB, PIK3, and-catenin beta-1 expression levels. RASE1 inhibited 3T3-L1 adipocyte differentiation and lipid accumulation by downregulating adipogenesis- and lipogenesis-promoting genes. RASE1 oral administration reduced mouse body and body fat pad weight and blood cholesterol, TG, ALT, AST, glucose, insulin, and adipokine levels. RASE1 suppressed adipogenic and lipid metabolism gene expression in mouse adipose and liver tissues and enhanced AMP-activated protein kinase levels in liver and adipose tissues and in serum adiponectin. RASE1 suppressed the NF-κB pathway and proinflammatory cytokines IL-10, IL-6, and TNF-α levels in mice which involve inflammation and progression of obesity. The overall results indicate RASE1 is a potential therapeutic formulation and functional food for treating or preventing obesity and inflammation.

Asiatic acid inhibits angiogenesis and vascular permeability through the VEGF/VEGFR2 signaling pathway to inhibit the growth and metastasis of breast cancer in mice.

Anti-angiogenic medicines have been evaluated as anticancer therapies, however, their use remains limited in clinical practice due to associated adverse effects. Asiatic acid (AA) is known to have broad-spectrum anticancer properties, however, its effects on angiogenesis in breast cancer remain to be fully established. In this study, we analyzed the inhibitory effects of AA on angiogenesis using human umbilical vein endothelial cells (HUVECs) cultured in vitro and on the growth and metastasis of a subcutaneous breast cancer 4T1 tumor model and a lung metastasis model in vivo. AA significantly inhibited HUVECs proliferation, migration, and tube formation in vitro. In vivo, AA significantly reduced the microvascular density and blood vascular permeability in breast cancer tumors and inhibited growth and lung metastasis. AA inhibited the expression of vascular endothelial growth factor (VEGF) in HUVECs and subsequently downregulated the phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) and its downstream target proteins including ERK1/2, Src, and FAK. These results indicate that AA significantly inhibits angiogenesis and blood vessel permeability through the VEGF/VEGFR2 signal axis to inhibit the growth and metastasis of breast cancer. Our data strongly demonstrate the potential applications of AA in the treatment of breast cancer.

LncRNA NEAT1 Promote Inflammatory Responses in Coronary Slow Flow Through Regulating miR-148b-3p/ICAM-1 Axis.

BackgroundCoronary slow flow (CSF) is an angiographic phenomenon characterized by delayed coronary opacification with normal or near-normal epicardial coronary arteries. The pathogenesis of CSF is closely related to inflammatory response. Accumulating evidence shows that long non-coding RNAs (lncRNAs) play an important role in cardiovascular disease. However, the mechanism underlying the influence of the lncRNA nuclear enriched abundant transcripts 1 (NEAT1) on CSF is still unknown.Patients and MethodsForty CSF patients and forty control subjects were included in the study and underwent coronary angiography, Seattle angina questionnaire (SAQ) and echocardiography. The plasma levels of the inflammatory factors soluble intercellular adhesion molecule-1 (sICAM-1), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-α) were determined by ELISA. The expression levels of NEAT1, miR-148b-3p and ICAM-1 in cells were measured by qRT-PCR or Western blotting. Cell proliferation was measured by 5‐Ethynyl‐2ʹ‐deoxyuridine (EdU) and Cell Counting Kit-8 (CCK-8) assay. Cell apoptosis was detected by apoptosis assay. The relationship between NEAT1 and miR-148b-3p was verified by luciferase reporter gene assay, RNA immunoprecipitation (RIP) assay and avidin-biotin pull-down assay. The relationship between ICAM-1 and miR-148b-3p was verified by luciferase reporter gene assay and avidin-biotin pull-down assay.ResultsThis study showed that plasma sICAM-1, miR-148b-3p, and NEAT1 as independent predictors of a CSF diagnosis. Furthermore, plasma NEAT1 level showed superior diagnostic ability for CSF compared with sICAM-1 and miR-148b-3p. It was also shown that high expression of NEAT1 in oxygen-glucose deprivation (OGD)-treated human umbilical vein endothelial cells (HUVECs) functions as a competing endogenous RNA (ceRNA). By specifically binding miR-148b-3p, it weakened the negative regulatory effects of miR-148b-3p on the ICAM-1 target gene leading to upregulated expression of ICAM-1. This interaction was also shown to inhibit HUVEC proliferation and enhance apoptosis.ConclusionThis study demonstrated for the first time the important mechanism of action of the NEAT1/miR-148b-3p/ICAM-1 axis in the progression of CSF disease, and indicated the potential of NEAT1, miR-148b-3p, and ICAM-1 as a new target for the diagnosis and treatment of CSF.

Enhanced Autophagy in GAB1-Deficient Vascular Endothelial Cells Is Responsible for Atherosclerosis Progression.

Autophagy is a host machinery that controls cellular health. Dysfunction of autophagy is responsible for the pathogenesis of many human diseases that include atherosclerosis obliterans (ASO). Physiologically, host autophagy removes aging organelles and delays the formation of atherosclerotic plaque. However, in ischemia event, dysregulated autophagy can be induced to trigger autosis, leading to an inevitable cellular death. Grb2-associated binder 1 (GAB1) is a docking/scaffolding adaptor protein that regulates many cell processes including autophagy. Our study first reported that the protein expression of GAB1 significantly decreased in ASO. Mechanically, our results showed that inhibition of Akt (protein kinase B), the upstream of mTOR (mechanistic target of rapamycin), significantly enhanced autophagy by demonstrating the downregulation of p62/Sequestosome 1 expression and the upregulation of the ratio of LC3II/LC3I. Conversely, we found that the inhibition of ERK1/2 (extracellular signal-regulated kinases1/2), p38, and JNK (c-Jun N-terminal kinase) signaling pathway, respectively, significantly inhibited autophagy by demonstrating the upregulation of p62 expression and the downregulation of the ratio of LC3II/LC3I. Further, we demonstrated that knockdown of GAB1 significantly increased autophagy in HUVECs (human umbilical vein endothelial cells) via activation of MAPK (mitogen-activated protein kinase) pathways that include ERK1/2, p38, and JNK. Moreover, we found that knockdown of GAB1 profoundly inhibited HUVEC proliferation, migration, and tube formation. Taken together, this study first suggests that GAB1 is a key regulator of autophagy in HUVECs. Targeting GAB1 may serve as a potential strategy for the atherosclerosis treatment.

EGCG Regulates Cell Apoptosis of Human Umbilical Vein Endothelial Cells Grown on 316L Stainless Steel for Stent Implantation.

BackgroundNowadays, medical grade 316L stainless steel (316L SS) is being widely used for intravascular stents, and the drug-eluting stent (DES) system is able to significantly reduce the occurrences of in-stent restenosis. But the drugs and the polymer coating used in DES potentially induce the forming of late stent thrombosis. In order to reduce the occurrence of ISR after stent implantation, the development of novel drugs for DESs is urgently needed.MethodsThis study aimed to investigate the potential mechanisms of epigallocatechin-3-gallate (EGCG) on human umbilical vein endothelial cells (HUVEC) grown on 316L stainless steel (316L SS) using flow cytometry and Q-PCR methods.ResultsOur results showed that EGCG (12.5, 25, 50, 100 μmol/L) significantly inhibited HUVEC proliferation. Flow cytometry analysis indicated that EGCG (25, 50, 100 μmol/L) induced apoptosis. Moreover, qRT-PCRrevealed that genes associated with cell apoptosis (caspase-3, 8, 9, Fas) and autophagy (Atg 5, Atg 7, Atg 12) were up-regulated after EGCG treatment.ConclusionThese findings indicate that EGCG possesses chemo preventive potential in stent coating which may serve as a novel new drug for stent implantation.

Robustaflavone induces G0/G1 cell cycle arrest and apoptosis in human umbilical vein endothelial cells and exhibits anti-angiogenic effects in vivo

We investigated the anti-angiogenic and pro-apoptotic effects of robustaflavone (RF), a naturally occurring biflavonoid, on human umbilical vein endothelial cells (HUVECs). RF inhibited HUVEC proliferation and showed cytotoxicity that inhibited HUVEC viability. RF-induced apoptosis was characterized by flow cytometry and caspase 3 analysis. We found that RF increased the number of sub-G1 cells and terminal deoxynucleotidyl transferase dUTP nick end-labeled cells. Additionally, RF induced caspase 3 and poly (ADP-ribose) polymerase activation. Potential molecular targets were identified using a human apoptosis antibody array. RF upregulated Bax, Bad, cleaved caspase 3, p21, and phosphorylated p53 levels. RF induced mitochondrial membrane potential loss and the release of cytochrome c and apoptosis-inducing factor. Cell cycle arrest at G0/G1 phase and the downregulation of Cdk4, Cdk6, and cyclin D1 expression were induced by RF. In vivo anti-angiogenic effects were investigated using a tumor allograft animal model and a Matrigel plug assay. RF reduced the volumes and weights of CT-26 cell-derived tumors. The blood vessel density was significantly decreased in RF-treated tumors. RF also inhibited VEGF-A-stimulated blood vessel formation in vivo in Matrigel plugs. These results suggest that RF can potentially inhibit angiogenesis-dependent tumor growth and metastasis.

Interfering Human Papillomavirus E6/E7 Oncogenes in Cervical Cancer Cells Inhibits the Angiogenesis of Vascular Endothelial Cells via Increasing miR-377 in Cervical Cancer Cell-Derived Microvesicles.

BackgroundThe dysregulation of the human papillomavirus 18 E6 and E7 oncogenes plays a critical role in the angiogenesis of cervical cancer (CC), including the proliferation, migration, and tube formation of vascular endothelial cells. Interfering E6/E7 increases the number of CC cell-derived microvesicles (CC-MVs). Additionally, microRNAs (miRNAs) can modulate CC angiogenesis and can be encapsulated in MVs.ObjectiveWe aim to investigate whether E6/E7 affects CC angiogenesis via regulating miRNAs in CC-MVs.MethodsCC-MVs were isolated from a CC cell line (HeLa) which were transfected with small interfering RNAs (siRNAs) against E6/E7 or co-transfected with miR-377 mimics/inhibitors. The expression of several miRNAs in CC-MVs was detected using quantitative real-time PCR. After co-incubating CC-MVs with human umbilical vein endothelial cells (HUVECs), cell proliferation, migration, and tube formation of HUVECs were determined using cell counting kit-8, transwell, and tube formation assays, respectively.ResultsMiR-377 was increased in E6/E7-interfering CC-MVs. Overexpressing miR-377 in CC-MVs suppressed HUVEC proliferation, migration, and tube formation. LPAR2, the cell surface G protein-coupled receptor, was the downstream target of miR-377 in HUVECs. The co-transfection of E6/E7 siRNAs and miR-377 inhibitors in CCs negated the effect of E6/E7 siRNAs on the elevation of miR-377 in CC-MVs. In HUVECs, the co-transfection of E6/E7 siRNAs and miR-377 inhibitors restored the LPAR2 expression which was reduced by the E6/E7 siRNA transfection. Meanwhile, miR-377 mimic reduced LPAR2 expression and inhibited HUVEC proliferation, migration, and tube formation, while such response was negated by LPAR2 overexpression.ConclusionInterfering E6/E7 increased miR-377 in CC-MVs, and overexpressing miR-377 in CC-MVs inhibited angiogenesis of HUVECs via reducing LPAR2.

Human cytomegalovirus promoting endothelial cell proliferation by targeting regulator of G-protein signaling 5 hypermethylation and downregulation

Interactions between human cytomegalovirus (HCMV) infection and environmental factors can increase susceptibility to essential hypertension (EH). Although endothelial dysfunction is the initial factor of EH, the epigenetic mechanisms through which HCMV infection induces endothelial cell dysfunction are poorly understood. Here, we evaluated whether HCMV regulated endothelial cell function and assessed the underlying mechanisms. Microarray analysis in human umbilical vein endothelial cells (HUVECs) treated with HCMV AD169 strain in the presence of hyperglycemia and hyperlipidemia revealed differential expression of genes involved in hypertension. Further analyses validated that the regulator of G-protein signaling 5 (RGS5) gene was downregulated in infected HUVECs and showed that HCMV infection promoted HUVEC proliferation, whereas hyperglycemia and hyperlipidemia inhibited HUVEC proliferation. Additionally, treatment with decitabine (DAC) and RGS5 reversed the effects of HCMV infection on HUVEC proliferation, but not triggered by hyperglycemia and hyperlipidemia. In summary, upregulation of RGS5 may be a promising treatment for preventing HCMV-induced hypertension.

Inhibitory Effects of Pectinase-Treated Prunus Mume Fruit Concentrate on Colorectal Cancer Proliferation and Angiogenesis of Endothelial Cells.

Pectinase is a well-known enzyme used in the food processing industry to produce fruit juice and concentrate. This study evaluated the anticancer and antiangiogenesis activities of pectinase-treated Prunus mume fruit concentrate (PC) and its phenolic components. PC treatment (250 to 1,000µg/mL) resulted in decreased proliferation of SW480 human colorectal cancer cells through S-phase cell cycle arrest; however, equivalent concentrations of PC did not show toxicity toward CRL-1539 colon normal cells. Furthermore, PC-induced caspase-dependent apoptosis in SW480 cells, which was characterized by accumulation of apoptotic cell population, cell shrinkage, formation of apoptotic bodies, upregulation of proapoptotic Bax, cleaved PARP, caspase-3, caspase-8, and caspase-9, and downregulation of antiapoptotic Bcl-2. Antiangiogenesis effects of PC were assessed using human umbilical vein endothelial cells (HUVECs). We found that PC did not inhibit HUVECs proliferation at concentrations of 500 to 1,500µg/mL. In addition, treatment with PC at nontoxic concentrations (500 to 1,000µg/mL) blocked vascular endothelial growth factor induced cell migration, invasion, capillary-like tube formation, and angiogenesis from rat aortic rings. HPLC-PDA analysis showed that there were at least four different phenolics including 5-HMF, neochlorogenic acid, protocatechuic acid, and syringic acid. Taken together, these results indicated that PC could be used as a good source of phenolic compounds with selective anticancer and antiangiogenesis activities. PRACTICAL APPLICATION: Pectinases are one of the well-known enzyme used in the part of food processing. Treatment of pectinase is a useful strategy to reduce viscosity, turbidity, and pulp particles in the production of fruit juice, extract, and concentrate. In the present study, we found that pectinase-treated P. mume fruit concentrate significantly suppresses colorectal cancer proliferation and angiogenesis of human umbilical vein endothelial cells. The significance of our findings is that pectinase-treated P. mume concentrate may be used as a commercial functional food material to inhibit colorectal cancer and angiogenesis.

Low Long Noncoding RNA Growth Arrest-Specific Transcript 5 Expression in the Exosomes of Lung Cancer Cells Promotes Tumor Angiogenesis.

Angiogenesis plays a key role in the development and progression of lung cancer. Recent studies have found that tumor cells can stimulate angiogenesis by secreting exosomes, which contain many long noncoding RNAs (lncRNAs), some of which are important for the development of lung cancer. However, the roles and mechanisms of exosomal lncRNAs in lung cancer angiogenesis have not yet been reported. In this study, lung cancer in mice was induced by urethane; we found that growth arrest specific 5 (GAS5) was lowly expressed in the serum exosomes and lung cancer tissues of mice with lung cancer. And there was a significant positive correlation between GAS5 expression in serum exosomes and lung cancer tissues. Furthermore, GAS5 was lowly expressed in human lung cancer tissues, lung cancer cells, and cells culture supernatant exosomes. The exosomes of lung cancer cells promoted human umbilical vein endothelial cells (HUVECs) proliferation and tube formation and inhibited their apoptosis. GAS5 overexpression in lung cancer cells increased GAS5 level in cell culture supernatant exosomes. And the exosomes of lung cancer cells containing high GAS5 level inhibited HUVECs proliferation and tube formation and increased their apoptosis. In addition, we found that GAS5 competitively bound miRNA-29-3p with phosphatase and tensin homolog (PTEN), upregulating PTEN mRNA and protein expression, and inhibited level of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PI3K) and serine/threonine kinase 1 (AKT) phosphorylation in HUVECs. Overall, our results suggest that exosomal GAS5 could be a new therapeutic target for lung cancer which inhibits angiogenesis.

CP‐25 suppressed the synovial pannus formation in experimental arthritis through Angiotensin □ and its signals

ObjectRheumatoid arthritis (RA) is a chronic systemic, autoimmune and inflammatory disease represented as synovitis, pannus formation, adjacent bone erosions, and joint destruction. Paeoniflorin‐6′‐O‐benzene sulfonate (code: CP‐25), a novel ester derivative of paeoniflorin, was evaluated the effects of anti‐angiopoiesis in rats with collagen‐induced arthritis (CIA) and regulated Angiotensin □(Ang□) and its signals pathway on vein endothelial cells.MethodsCIA rats were treated with CP‐25 (50 mg/kg) from days 14 to 35 after immunization. The joint synovial hemodynamics of rats were detected by ultrasonographic methodology and the synovial pannus formation was observed by histopathologic assay. To detect the role of CP‐25 on the synovial pannus formation, human umbilical vein endothelial cells (HUVECs) were co‐stimulated with Ang □ (10−7 mol/L) and TNF‐α (5 ng/ml) in vitro, and CP‐25(10−5 mol/L), Losartan (10−5 mol/L, AT1R blocker) or PD123319 (10−5 mol/L, AT2R blocker) were individual or combined treatment. The effects of CP‐25 on HUVECs functions such as proliferation, migration were detected by Cell Counting Kit‐8 assay (CCK8) or transwell assay. The tube formation were examined by using Matrigel model. The protein expression of AT1R/AT2R signaling pathways in HUVECs were analyzed by western blot.ResultsCP‐25 could alleviate pathological changes and inhibit the synovial pannus formation in CIA rats, decrease regional synovial blood flow signals in the local swelling knee joint. CP‐25 could inhibit HUVEC proliferation, migration as well as tube formation which co‐stimulated with Ang□ and TNF‐α in vitro. CP‐25 showed a synergistic role with Lostan (AT1R antagonist) on down‐regulated the expression of GRK2, p‐ERK1/2, p‐IκBα and p‐P65. Meanwhile, CP‐25 could also inhibit the high level of GRK2, p‐ERK1/2, p‐IκBα and p‐P65 which induced by AT2R antagonist (PD123319).ConclusionCP‐25 could present a potent anti‐angiopoiesis effect in CIA rats and inhibit proliferation, migration as well as tube formation function of the HUVEC through Ang□ and its signals mediating AT1R/AT2R‐GRK2‐ERK1/2‐NF‐κB pathway.Support or Funding InformationThis research was supported by the National Natural Science Foundation of China (No. 81330081; 81673444)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

MicroRNA-34a promotes hypertension-induced vascular injury by targeting B-lymphocytoma-2

Objective To investigate the level of microRNA (miRNA, miR)-34a in serum of hypertensive patients and its effect on proliferation and apoptosis of human umbilical vein endothelial cells (HUVECs) by targeting B cell lymphoma/leukemia-2 (bcl-2), and clarify the underlying mechanism of hypertension-induced vascular injury. Methods Real-time quantitative polymerase chain reaction (qPCR) was used to detect the level of miR-34a in serum of 98 hypertensive patients and 65 healthy controls; the potential targets of miR-34a was predicted by TargetScan; the interaction between miR-34a and bcl-2 was measured by dual luciferase reporter assay, western blot and qPCR; the effect of miR-34a on proliferation and apoptosis of HUVECs was detected by methyl thiazol tetrazolium (MTT) and flow cytometry. Results The level of miR-34a in serum of 98 hypertensive patients (2.432±0.201) was significantly higher than that of 65 healthy controls (0.996±0.112, P 0.05). The protein and mRNA level of bcl-2 in HUVECs after overexpressing miR-34a (0.403±0.041, 0.206±0.018) decreased significantly than that in Blank group (1.000±0.098, 1.000±0.067) and miR-NC group (0.996±0.087, 1.012±0.049). The proliferation of HUVECs after overexpressing miR-34a (48 h: 0.269±0.021, 72 h: 0.324±0.028) decreased significantly than that of the Blank group (48 h: 0.324±0.025, 72 h: 0.458±0.044) and miR-NC group (48 h: 0.335±0.028, 72 h: 0.469±0.051, P<0.05), and apoptosis (23.54±2.65) was significantly higher than that of Blank (6.45±0.42) and miR-NC (7.32±0.54, P<0.05). Conclusion MiR-34a is overexpressed in serum of hypertensive patients. Overexpressing miR-34a can inhibit proliferation of HUVECs by targeting bcl-2 expression, which may be one of the potential mechanisms of vascular injury in hypertensive patients and provide experimental basis for using miR-34a as a new target to diagnose and treat hypertension in the future. Key words: MicroRNA-34a; B cell lymphoma/leukemia-2; Hypertension; Vascular injury

Bromophenol curcumin analog BCA-5 exerts an antiangiogenic effect through the HIF-1α/VEGF/Akt signaling pathway in human umbilical vein endothelial cells.

The bromophenol curcumin analog 1,5-bis(3-bromo-4, 5-dimethoxyphenyl) penta-1, 4-dien-3-one (BCA-5) was assayed for antiangiogenic activity. Tandem mass tag labeling and liquid chromatography-tandem mass spectrometry were used to quantify the dynamic changes in the human umbilical vein endothelial cell (HUVEC) proteome. Functional annotation showed that BCA-5 might inhibit compounds related to the extracellular matrix, compounds that possess cytoskeletal protein-binding activity, and compounds that interact with cell motility-related enzymes, indicating antiangiogenic potential. In-vitro experiments have shown that BCA-5 inhibited HUVEC proliferation and induced HUVEC apoptosis. BCA-5 inhibited HUVEC migration, invasion, and tubular formation. BCA-5 decreased the phosphorylation of Akt and endothelial nitric oxide synthase; it also reduced the expression of hypoxia-inducible factor-1α and vascular endothelial cell growth factor in a dose-dependent manner. These results suggest that BCA-5 has antiangiogenic properties and should be considered a potent antiangiogenesis drug for the treatment of cancer.

Melatonin Protects Against Hypoxia/Reoxygenation-Induced Dysfunction of Human Umbilical Vein Endothelial Cells Through Inhibiting Reactive Oxygen Species Generation.

Hypoxia/reoxygenation (H/R) in human umbilical vein endothelial cells (HUVECs) induces oxidative stress and eventually leads to vascular injury. The aim of this study was to examine the effect of melatonin on HUVECs injured by H/R and explore the underlying mechanisms. A model of HUVECs under hypoxia/reoxygenation was established. Cell migration and adhesive ability was measured by wound healing and adhesion assays. Cell proliferation was measured by EdU assay. Production of reactive oxygen species (ROS) was evaluated by CM-H2DCFDA staining. Actin cytoskeleton rearrangement was examined by immunofluorescence. Western blotting analysis were used to analyze P38 and HSP27 phosphorylation levels. H/R inhibited HUVEC proliferation, cell migratory and adhesive capacities, whereas melatonin (1~100 μM) inhibited these effects in a dose-dependent manner. Melatonin alone did not affect HUVEC viability, however, it inhibited the increase in ROS production and cytoskeleton disruption elicited by H/R, and it dose-dependently prevented H/R-induced upregulation of P38 and HSP27 phosphorylation. In addition, the ROS scavenger N-acetyl-L-cysteine markedly inhibited increased phosphorylation levels of P38 and HSP27 under H/R. Melatonin may have a potential clinical effect in trials of H/R-induced vascular injury through its antioxidant property.

Nicotinic acid inhibits angiogenesis likely through cytoskeleton remodeling

ABSTRACTAngiogenesis is a physiological procedure during which the new blood vessels develop from the pre-existing vessels. Uncontrolled angiogenesis is related to various diseases including cancers. Clinical inhibition of undesired angiogenesis is still under investigation. We utilized nicotinic acid, a family member of the B-vitamin niacin (vitamin B3) that has been used in the prevention and treatment of atherosclerosis or other lipid-metabolic disorders, to treat human umbilical vein endothelial cells (HUVECs) and chick chorioallantoic membrane (CAM), and investigated its influence on angiogenesis in vitro and in vivo. We found that nicotinic acid could obviously inhibit HUVEC proliferation induced by vascular endothelial growth factor. Both the in vitro and in vivo assays showed that nicotinic acid could significantly inhibit the process of angiogenesis. To further investigate the mechanism underlying the effect of nicotinic acid on angiogenesis, we found that it might function via regulating the cytoskeleton arrangements, especially the rearranging the structures of F-actin and paxillin. In summary, we discovered that nicotinic acid could obviously inhibit the process of angiogenesis by changing the angiogenesis factor expression levels and inducing the cytoskeleton rearrangement of endothelial cells.

The oxidized phospholipid POVPC impairs endothelial function and vasodilation via uncoupling endothelial nitric oxide synthase

Endothelial dysfunction is an early stage of atherosclerosis. We recently have shown that 25-hydroxycholesterol found in atherosclerotic lesions could impair endothelial function and vasodilation by uncoupling and inhibiting endothelial nitric oxide synthase (eNOS). 1-Palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), the oxidation product of oxidized low-density lipoprotein, is another proinflammatory lipid and has also been found in atherosclerotic lesions. However, whether POVPC promotes atherosclerosis like 25-hydroxycholesterol remains unclear. The purpose of this study was to explore the effects of POVPC on endothelial function and vasodilation. Human umbilical vein endothelial cells (HUVECs) were incubated with POVPC. Endothelial cell proliferation, migration and tube formation were measured. Nitric oxide (NO) production and superoxide anion generation (O2−) were determined. The expression and phosphorylation of endothelial nitric oxide synthase (eNOS), AKT, PKC-βII and P70S6K as well as the association of eNOS and heat shock protein 90 (HSP90) were detected by immunoblotting and immunoprecipitation. Endothelial cell apoptosis was monitored by TUNEL staining. The expression of Bcl-2, Bax, and Cleaved Caspase 3 were detected by immunoblotting. Finally, aortic ring from C57BL6 mice were isolated and treated with POVPC and the endothelium-dependent vasodilation was evaluated. POVPC significantly inhibited HUVECs proliferation, migration, tube formation, decreased NO production but increased O2− generation. POVPC inhibited the phosphorylation of Akt and eNOS at Ser1177, increased activation of PKC-βII, P70S6K and the phosphorylation of eNOS at Thr495, reduced the association of HSP90 with eNOS. Meanwhile, POVPC induced endothelial cell apoptosis by inhibiting Bcl-2 expression, increasing Bax and cleaved caspase-3 expressions as well as caspase-3 activity and impaired endothelium-dependent vasodilation. These data demonstrated that POVPC impaired endothelial function by uncoupling and inhibiting eNOS as well as by inducing endothelial cell apoptosis. Therefore, POVPC may play an important role in the development of atherosclerosis and may be considered as a potential therapeutic target for atherosclerosis.