Aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor is involved in regulation of many essential biological processes including vascular development and angiogenesis. 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) is an AhR ligand, which regulates immune responses and cancer cell growth. However, the roles of the ITE/AhR pathway in mediating placental angiogenesis remains elusive. Here, we determined if ITE affected placental angiogenic responses via AhR in human umbilical vein (HUVECs) and artery endothelial (HUAECs) cells in vitro. We observed that ITE dose- and time-dependently inhibited proliferation and viability of HUAECs and HUVECs, whereas it inhibited migration of HUAECs, but not HUVECs. While AhR siRNA significantly suppressed AhR protein expression in HUVECs and HUAECs, it attenuated the ITE-inhibited angiogenic responses of HUAECs, but not HUVECs. Collectively, ITE suppressed angiogenic responses of HUAECs and HUVECs, dependent and independent of AhR, respectively. These data suggest that ITE may regulate placental angiogenesis.

Placental angiogenesis is dramatically increased during pregnancy in association with the elevated placental blood flows to support the rapidly growing fetus. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an environmental toxicant and a ligand of aryl hydrocarbon receptor (AhR). Herein, we investigated the effects of TCDD on proliferation, migration, and viability of fetoplacental endothelial cells in response to a complete growth medium which contained serum and growth supplement using human umbilical cord vein (HUVECs) and artery (HUAECs) cells as models. We found that TCDD dose- and time-dependently inhibited (p < 0.05) proliferation of HUVECs and HUAECs. Treatment with TCDD at 10 nM for 6 days inhibited (p < 0.05) migration (by ∼ 30%) of HUAECs, but not HUVECs. TCDD at 10nM also decreased (p < 0.05) viability of HUVECs and HUAECs. Interestingly, specific AhR siRNA blocked (p < 0.05) the TCDD-inhibited cellular responses in HUAECs, but not HUVECs. Nonetheless, TCDD at 10nM neither affected the cell cycle progression, nor did it induce cell apoptosis in HUVECs and HUAECs. In addition, TCDD at 10 nM also did not alter activation of ERK1/2 and AKT1 in HUVECs and HUAECs. Collectively, TCDD suppresses proliferation and/or migration (two key steps of angiogenesis) of HUVECs and HUAECs independent and dependent of AhR, respectively. These data suggest that TCDD inhibited growth of HUVECs and HUAECs via decreasing cell viability. Thus, TCDD may inhibit fetoplacental angiogenesis, leading to negative pregnancy outcomes.

Hypoxia-induced upregulation of lincRNA-p21 in tumor tissue was previously shown by our group to be related to poor prognosis in resected non-small cell lung cancer (NSCLC) patients. In the present study, we have evaluated the presence of lincRNA-p21 in extracellular vesicles (EVs) from NSCLC patients and assessed its potential as a prognostic biomarker. High EV lincRNA-p21 levels in blood from the tumor-draining vein were associated with shorter time to relapse and shorter overall survival. Moreover, the multivariate analysis identified high lincRNA-p21 levels as an independent prognostic marker. In addition, lincRNA-p21 was overexpressed in H23 and HCC44 NSCLC cell lines and their derived EVs under hypoxic conditions. Functional assays using human umbilical vein endothelial cells (HUVECs) showed that tumor-derived EVs enriched in lincRNA-p21 affected endothelial cells by promoting tube formation and enhancing tumor cell adhesion to endothelial cells. Additionally, the analysis of selected EV microRNAs related to angiogenesis and metastasis showed that the microRNAs correlated with EV lincRNA-p21 levels in both patients and cell lines. Finally, EV co-culture with HUVEC cells increased the expression of microRNAs and genes related to endothelial cell activation. In conclusion, EV lincRNA-p21 acts as a novel prognosis marker in resected NSCLC patients, promoting angiogenesis and metastasis.

BACKGROUND Endothelial cells are involved in vascular homeostasis, and endothelial cell dysfunction is involved in the pathogenesis of cardiovascular disease. This study aimed to investigate the effects of microRNA-154 in human umbilical vein endothelial cells (HUVECs) following injury induced by hydrogen peroxide (H₂O₂). MATERIAL AND METHODS Cell viability and apoptosis of HUVECs treated with H₂O₂ were measured. The expression of microRNA-154 was detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell survival, caspase-3 activity, and the apoptosis rate were evaluated in H₂O₂-treated HUVECs cells after the upregulation and down-regulation of microRNA-154 expression. The interaction between microRNA-154 and Dickkopf WNT signaling pathway inhibitor 2 (DKK2) was predicted by bioinformatics analysis and was verified by luciferase reporter gene assay and Western blot. The effects of DKK2 short-interfering RNA (siRNA) on antioxidant injury in HUVECs cells were determined. RESULTS The survival rate of HUVECs exposed to H₂O₂ was significantly reduced and the apoptosis rate was significantly increased, and H₂O₂ significantly inhibited the expression of microRNA-154 in a dose-dependent manner. Overexpression of microRNA-154 increased cell survival, reduced the activity of caspase-3, and reduced cell apoptosis. Inhibition of microRNA-154 expression decreased cell survival, increased the activity of caspase-3, and promoted cell apoptosis. Luciferase reporter gene assay and Western blot showed that microRNA-154 interacted with the Wnt pathway molecule DKK2 in HUVECS. Also, DDK2 siRNA resulted in a similar protective effect on H₂O₂-treated HUVECs as overexpression of microRNA-154. CONCLUSIONS Oxidative injury in HUVECs was regulated by microRNA-154 targeting the Wnt/ß-catenin signaling pathway.

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.

The mechanisms of intervertebral disc degeneration (IDD) involve numerous factors, including loss of the extracellular matrix (ECM) and vascular ingrowth. Melatonin has been reported to protect intervertebral discs (IVDs) from degeneration and to exert a potential anti-angiogenic effect. The aim of the present study was to investigate the anti-angiogenic and anabolic effects of melatonin in IVDs. Human nucleus pulposus (NP) and degenerative nucleus pulposus (DNP) cells were isolated and treated with melatonin. The results indicated that melatonin promoted ECM synthesis and NP cell proliferation. In addition, an NP/DNP and human umbilical vein endothelial cell (HUVEC) co-culture model was used to investigate the anti-angiogenesis effect of melatonin. Melatonin was indicated to suppress tube formation and migration of HUVECs in culture with NP cell-conditioned medium, as well as in an NP cell co-culture model. Fluorescence-labeled vascular endothelial growth factor (VEGF) was used to study the binding between VEGF and its receptor. The results of the present study indicated that melatonin exerts an angiogenic effect via inhibition of the binding of VEGF to its receptor in HUVECs. Taken together, these results suggest that melatonin is a potential agent to prevent IDD.

Endothelial-to-mesenchymal transition (EndMT), the process by which an endothelial cell (EC) undergoes a series of molecular events that result in a mesenchymal cell phenotype, plays an important role in atherosclerosis. 1-Palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), derived from the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphatidylcholine, is a proinflammatory lipid found in atherosclerotic lesions. Whether POVPC promotes EndMT and how simvastatin influences POVPC-mediated EndMT remains unclear. Here, we treated human umbilical vein ECs with POVPC, simvastatin, or both, and determined their effect on EC viability, morphology, tube formation, proliferation, and generation of NO and superoxide anion (O2•-). Expression of specific endothelial and mesenchymal markers was detected by immunofluorescence and immunoblotting. POVPC did not affect EC viability but altered cellular morphology from cobblestone-like ECs to a spindle-like mesenchymal cell morphology. POVPC increased O2- generation and expression of alpha-smooth muscle actin, vimentin, Snail-1, Twist-1, transforming growth factor-beta (TGF-β), TGF-β receptor II, p-Smad2/3, and Smad2/3. POVPC also decreased NO production and expression of CD31 and endothelial NO synthase. Simvastatin inhibited POVPC-mediated effects on cellular morphology, production of O2•- and NO, and expression of specific endothelial and mesenchymal markers. These data demonstrate that POVPC induces EndMT by increasing oxidative stress, which stimulates TGF-β/Smad signaling, leading to Snail-1 and Twist-1 activation. Simvastatin inhibited POVPC-induced EndMT by decreasing oxidative stress, suppressing TGF-β/Smad signaling, and inactivating Snail-1 and Twist-1. Our findings reveal a novel mechanism of atherosclerosis that can be inhibited by simvastatin.

Atherosclerosis is an inflammatory disease of the arterial wall, which involves endothelial cells and immune cells. Endothelial dysfunction has been considered an important step in the initiation of the disease. TIPE1 is a newly identified protein of the TIPE family, and plays a vital role in inflammation and tumorigenesis. However, its role in atherogenesis remains unclear. In this study, we demonstrated that TIPE1 promoted atherogenesis by inducing endothelial dysfunction. When human umbilical vein endothelial cells (HUVECs) were exposed to oxidative stress, the level of TIPE1 was significantly up-regulated, and the ROS generation markedly increased in TIPE1 over-expressing HUVECs. As a result, the growth of HUVECs was inhibited, and the apoptosis was enhanced. However, the cell contact ability between HUVECs and THP-1 cells were augmented due to the up-regulation of adhesion molecules such as E-selectin and ICAM-1 induced by TIPE1 overexpression. Importantly, ApoE-/- mice injected with TIPE1 recombinant lentivirus developed significantly severe atherosclerosis accompanied by hyperglycemia, hypercholesterolemia and increased white blood count. These findings indicated that excessive ROS induced by the overexpression of TIPE1 in endothelial cells accelerated the process of atherogenesis.

Fetoplacental endothelial cells reside under physiological normoxic conditions (∼2-8% O2) in vivo. Under such conditions, cells are believed to sense O2 changes primarily via hypoxia inducible factor 1 α (HIF1A). However, little is known regarding the role of HIF1A in fetoplacental endothelial function under physiological normoxia. We recently reported that physiological chronic normoxia (PCN; 20-25 day, 3% O2) enhanced FGF2- and VEGFA-stimulated proliferation and migration of human umbilical vein endothelial cells (HUVECs) via the MEK/ERK1/2 and PI3K/AKT1 pathways compared to standard cell culture normoxia (SCN; ambient O2: ∼21% O2). Here, we investigated the action of HIF1A in regulating these cellular responses in HUVECs. HIF1A adenovirus infection in SCN-cells increased HIF1A protein expression, enhanced FGF2- and VEGFA-stimulated cell proliferation by 2.4 and 2.0-fold respectively, and promoted VEGFA-stimulated cell migration by 1.4-fold. HIF1A adenovirus infection in SCN-cells did not affect either basal or FGF2- and VEGFA-induced ERK1/2 activation, but it decreased basal AKT1 phosphorylation. Interestingly, HIF1A knockdown in PCN-cells via specific HIF1A siRNA transfection did not alter FGF2- and VEGFA-stimulated cell proliferation and migration, or ERK1/2 activation; however, it inhibited FGF2-induced AKT1 activation by ∼50%. These data indicate that HIF1A differentially regulates cell proliferation and migration, and ERK1/2 and AKT1 activation in PCN- and SCN-HUVECs. These data also suggest that HIF1A critically regulates cell proliferation and migration in SCN-, but not in PCN-HUVECs.

Angiogenesis is a well-established target in anti-cancer therapy. Although vascular endothelial growth factor (VEGF)-mediated angiogenesis apparently requires the Rho GTPases Rac1 and Cdc42, the relevant mechanisms are unclear. Here, we determined that activated Rac1/Cdc42 in MCF-7 breast cancer cells could decrease p53 protein levels and increase VEGF secretion to promote proliferation and tube formation of human umbilical vein endothelial cells (HUVECs). However, these effects are reversed after ubiquitin-proteasome breakage. In exploring potential mechanisms for this relationship, we confirmed that activated Rac1/Cdc42 could enhance p53 protein ubiquitination and weaken p53 protein stability to increase VEGF expression. Furthermore, in a xenograft model using nude mice that stably express active Rac1/Cdc42 protein, active Rac1/Cdc42 decreased p53 levels and increased VEGF expression. Additionally, tumor angiogenesis was inhibited, and p53 protein levels were augmented, by intratumoral injection of the ubiquitin-proteasome inhibitor MG132. Finally in 339 human breast cancer tissues, our analyses indicated that Rac1/Cdc42 expression was related to advanced TNM staging, high proliferation index, ER status, and positive invasive features. In particular, our data suggests that high Rac1/Cdc42 expression is correlated with low wt-p53 and high VEGF expression. We conclude that activated Rac1/Cdc42 is a vascular regulator of tumor angiogenesis and that it may reduce stability of the p53 protein to promote VEGF expression by enhancing p53 protein ubiquitin.

The use of angiotensin II inhibitors is associated with a low risk of recurrence and metastasis in hepatocellular carcinoma (HCC) patients. Vascular cell adhesion molecule-1 (VCAM-1) is a key factor in tumor metastasis.

Circular RNAs (circRNAs) are a group of non-protein-coding RNAs generated from back splicing. Emerging evidence has demonstrated its vital regulation on angiogenesis. However, the underlying mechanism responsible for circRNAs effects on vascular endothelial cells is still unclear. In the present study, we screened the expression profiles and investigated the physiological role of circRNAs in hypoxia-induced human umbilical vein endothelial cells (HUVECs). Using circRNA microarray analysis, we identified 36 circRNAs that were significantly dysregulated including 14 down-regulated circRNAs and 22 up-regulated with 2-fold change (P < 0.05). From the over-expressed circRNAs, hsa_circ_0010729 was selected as candidate circRNA and which was validated to be significantly up-regulated using RT-PCR. In loss-of-function experiments of HUVECs, hsa_circ_0010729 knockdown suppressed the proliferation and migration ability and enhanced apoptosis. Bioinformatic prediction and luciferase assay revealed that hsa_circ_0010729 and hypoxia inducible factor 1 alpha (HIF-1α) were targeted by miR-186. Validation experiments verified that hsa_circ_0010729 was co-expressed with HIF-1α, being negatively correlated with miR-186. Moreover, rescue experiments demonstrated that miR-186 inhibitor could reverse the role of hsa_circ_0010729 knockdown on HUVECs progression. Overall, the present study identifies the crucial regulation of hsa_circ_0010729 on vascular endothelial cell proliferation and apoptosis via targeting miR-186/HIF-1α axis.

The apolipoprotein A-I mimetic peptide D-4F, among its anti-atherosclerotic effects, improves vasodilation through mechanisms not fully elucidated yet.

Atherosclerosis (AS) is the leading cause of cardiovascular disease. Circular RNA hsa_circ_0003204 (hsa_circ_0003204) was elevated in oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells. However, the role and molecular mechanism of hsa_circ_0003204 in the AS process have not been studied.

Astragalus membranaceus (AM) is a traditional Chinese medicinal herb, whose cytoprotective effects remain largely unknown. Here, the bacterial endotoxin lipopolysaccharide (LPS) was applied to a human pulmonary type II‑like epithelial lung adenocarcinoma cell line, a human umbilical vein endothelial cell line, and a human bladder carcinoma cell line to construct in vitro models of intracellular oxidative stress. The authors assayed the cellular and mitochondrial cytoprotective effects of varying doses of AM root extract upon these cell lines. The cell lines were cultured as follows: LPS‑only group, four LPS+AM groups treated with various AM concentrations plus LPS, and an untreated control group. Flow cytometry was used to assess apoptosis and cell cycle progression. A 2',7'‑dichlorofluorescein‑diacetate assay was used to quantitate reactive oxygen species (ROS) generation. Mitochondrial membrane potential (Δψmit) was analyzed by Rhodamine 123 assay. Western blotting was performed to detect cleaved caspase‑3, p53, and B cell lymphoma (Bcl)‑2 levels. Across all cell lines, LPS significantly elevated apoptosis rates, shifted cells to S/G2 phase, increased ROS production, reduced Δψmit, upregulated cleaved caspase‑3, upregulated p53, and downregulated Bcl‑2 relative to controls (all P<0.05). As a general trend, increasing AM concentrations produced progressively greater reductions in the apoptosis rate, greater reductions in S/G2 phase %, greater reductions in ROS production, greater increases in Δψmit, greater reductions in cleaved caspase‑3 and p53 expression, and greater increases in Bcl‑2 expression. AM treatment protects human pulmonary and bladder epithelial cells, in addition to human endothelial cells, from LPS‑induced apoptosis, in a dose‑dependent manner.

Tumor angiogenesis is an important factor which precipitates recurrence and metastasis of colorectal cancer (CRC). Angiogenesis is also a significant feature which accompanies invasion and metastasis of CRC. Tumor hypoxia activates hypoxia inducible factor (HIF), which promotes angiogenesis in CRC. HIF significantly promotes cell proliferation and angiogenesis in CRC, facilitating invasion and metastasis. Tanshinone IIA (Tan IIA) has been revealed to effectively inhibit angiogenesis in CRC, although the underlying mechanism remains to be determined. The aim of the present study was to determine the effects of HIF‑1α on hypoxia induced angiogenesis in CRC cells, the effects of Tan IIA on the expression of pro‑angiogenic factors in CRC cells, and on human umbilical vein endothelial cell (HUVEC) tube formation in normal and hypoxic conditions. The results of the present study revealed that Tan IIA not only decreased HIF‑1α expression and inhibited the secretion level of vascular endothelial growth factor and basic fibroblast growth factor, but also efficiently decreased proliferation, tube formation and metastasis of HUVECs. The results highlight the potential of Tan IIA‑mediated targeting of HIF‑1α as a potential therapeutic option for treatment of patients with CRC.

Cadmium (Cd) is a heavy metal and environmental toxin. Exposure to Cd has been associated with a variety of human cancers. In this study, we performed in vitro assays to examine the effects of cadmium chloride (CdCl₂) on A549 cells, a human lung adenocarcinoma cell line. Cd does not affect proliferation, migration, or apoptosis of A549 cells at concentrations of 0.1-10 μM. At 0.5 and 1 μM, Cd increases the expression of vascular endothelial growth factor (VEGF) (p < 0.05, p < 0.01, respectively), but not basic fibroblast growth factor (b-FGF) in A549 cells. The conditioned media were collected from the A549 cells treated with 1 μM Cd and were co-cultured with human umbilical vein endothelial cells (HUVECs). Upon treatment with the conditioned media, the proliferation and migration of HUVECs significantly increased (p < 0.01, p < 0.05, respectively), while apoptosis remained unchanged. In addition, 1 μM Cd increases the level of hypoxia inducible factor 1-α (HIF1-α), which is a positive regulator of VEGF expression. Although low-dose Cd does not directly affect the growth of lung adenocarcinoma cells, it might facilitate the development of tumors through its pro-angiogenic effects.

Corneal neovascularization (angiogenesis and lymphangiogenesis) compromises corneal transparency and transplant survival, however, the molecular mechanisms of corneal host epithelial and stromal cells in neovascularization have not yet been fully elucidated. Furthermore, the contribution and mechanism of corneal host endothelial cells involved in neovascularization are largely unexplored.

Mesenchymal stem cells (MSCs) have emerged as putative therapeutic tools due to their intrinsic tumor tropism, and anti-tumor and immunoregulatory properties. The limited passage and self-differentiation abilities of MSCs in vitro hinder preclinical studies on them. In this study, we focused on the safety of immortalized mesenchymal stem cells (im-MSCs) and, for the first time, studied the feasibility of im-MSCs as candidates for the treatment of glioma. The im-MSCs were constructed by lentiviral transfection of genes. The proliferative capacity of im-MSCs and the proliferative phenotype of MSCs and MSCs co-cultured with glioma cells (U87) were measured using CCK-8 or EdU assays. After long-term culture, karyotyping of im-MSCs was conducted. The tumorigenicity of engineered MSCs was evaluated using soft agar cloning assays. Next, the engineered cells were injected into the brain of female BALB/c nude mice. Finally, the cell membranes of im-MSCs were labeled with DiO or DiR to detect their ability to be taken up by glioma cells and target in situ gliomas using the IVIS system. Engineered cells retained the immunophenotype of MSC; im-MSCs maintained the ability to differentiate into mesenchymal lineages in vitro; and im-MSCs showed stronger proliferative capacity than unengineered MSCs but without colony formation in soft agar, no tumorigenicity in the brain, and normal chromosomes. MSCs or im-MSCs co-cultured with U87 cells showed enhanced proliferation ability, but did not show malignant characteristics in vitro. Immortalized cells continued to express homing molecules. The cell membranes of im-MSCs were taken up by glioma cells and targeted in situ gliomas in vivo, suggesting that im-MSCs and their plasma membranes can be used as natural drug carriers for targeting gliomas, and providing a safe, adequate, quality-controlled, and continuous source for the treatment of gliomas based on whole-cell or cell membrane carriers.

Chronic wounds resulting from diabetes are a major health concern in both industrialized and developing countries, representing one of the leading causes of disability and death. This study aimed to investigate the effect of adipose mesenchymal stem cell-derived exosomes (ADSC-exos) on diabetic wounds and the mechanism underlying this effect. The results showed that ADSC-exos could improve oxidative stress and secretion of inflammatory cytokines in diabetic wounds, thereby increasing periwound vascularization and accelerating wound healing. At the cellular level, ADSC-exos reduced reactive oxygen species (ROS) generation in human umbilical vein endothelial cells (HUVECs) and improved mitochondrial function in a high-glucose environment. Moreover, the Western blot analysis showed that the high-glucose environment decreased Sirtuin 3 (SIRT3) expression, while exosome treatment increased SIRT3 expression. The activity of superoxide dismutase 2 (SOD2) was enhanced, and the level of inflammatory cytokines was decreased. Further, SIRT3 interference experiments indicated that the effects of ADSC-exos on oxidative stress and angiogenesis were partly dependent on SIRT3. After SIRT3 was inhibited, ROS production increased, while mitochondrial membrane potential and SOD2 activity decreased. These findings confirmed that ADSC-exos could improve the level of high-glucose-induced oxidative stress, promote angiogenesis, and reduce mitochondrial functional impairment and the inflammatory response by regulating SIRT3/SOD2, thus promoting diabetic wound healing.