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Recently, XRCC1 polymorphisms were reported to be associated with glioma in Chinese population. However, only a few studies reported on the XRCC1 expression, and cancer progression. In this study, we investigated whether XRCC1 plays a role in glioma pathogenesis. Using the tissue microarray technology, we found that XRCC1 expression is significantly decreased in glioma compared with tumor adjacent normal brain tissue (P < 0.01, χ 2 test) and reduced XRCC1 staining was associated with WHO stages (P < 0.05, χ 2 test). The mRNA and protein levels of XRCC1 were significantly downregulated in human primary glioma tissues (P < 0.001, χ 2 test). We also found that XRCC1 was significantly decreased in glioma cell lines compared to normal human astrocytes (P < 0.01, χ 2 test). Overexpression of XRCC1 dramatically reduced the proliferation and caused cessation of cell cycle. The reduced cell proliferation is due to G1 phase arrest as cyclin D1 is diminished whereas p16 is upregulated. We further demonstrated that XRCC1 overexpression suppressed the glioma cell migration and invasion abilities by targeting MMP-2. In addition, we also found that overexpression of XRCC1 sharply inhibited angiogenesis, which correlated with down-regulation of VEGF. The data indicate that XRCC1 may be a tumor suppressor involved in the progression of glioma.

Angiogenesis plays a vital role in the process of embryo implantation, as it improves endometrial receptivity and guides embryo implantation, thus creating a favorable environment for subsequent development of the embryo. Hence, a theory of achieving contraception by inhibiting angiogenesis was put forward. Here, we screened the drugs inhibiting angiogenesis using cell scratch wound assay and a 3D biomimetic vascular microfluidic chip, then observed the effect of them on contraception by injecting these drugs into fertilized mice and observing if the embryos were implanted. We preliminarily verify the feasibility of contraception by inhibiting angiogenesis and gives a new direction in the development of contraceptive pills., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Angiogenesis is recognized as a sign of cancer and facilitates cancer progression and metastasis. Suppression of angiogenesis is a desirable strategy for gastric cancer (GC) management. In this study, we showed a novel role of gastrin in angiogenesis of GC. We observed that treatment with gastrin 17 (G17) increased the proliferation of AGS cells and enhanced tube formation during normoxia and hypoxia. The expression level of VEGF were increased by G17 treatment as well. Experiments on the mechanism showed that G17 promoted HIF-1α expression, which subsequently enhanced β-catenin nuclear localization and activation of TCF3 and LEF1 and finally resulted in angiogenesis by upregulating VEGF. An in vivo experiment confirmed that G17 enhanced GC cell proliferation and angiogenesis in the resultant tumor. In conclusion, our findings indicate that gastrin promotes angiogenesis via activating HIF-1α/β-catenin/VEGF axis in GC., (Copyright © 2019. Published by Elsevier B.V.)

Galectin-3, a biomarker linking oxidative stress and inflammation, participates in different mechanisms related to atherothrombosis, such as inflammation, proliferation, or macrophage chemotaxis. Accumulating evidence indicates that galectin-3 may also promote atherogenesis through inducing endothelial dysfunction. Lectin-like oxidized low-density lipoprotein (oxLDL) receptor-1 (LOX-1), a receptor for oxLDL uptake, contributes to oxLDL-induced endothelial dysfunction. Whether galectin-3 induces endothelial dysfunction through modulation of LOX-1-mediated signaling remains unclear. In the present study, we explored the mechanisms underlying galectin-3 enhanced cytotoxicity of oxLDL in human umbilical vein endothelial cells (HUVECs) and the role of LOX-1. Incubation of HUVECs with galectin-3 increased the expression of LOX-1 in RNA and protein levels. In addition, the expression of LOX-1 induced by oxLDL was promoted by galectin-3. However, pretreatment of LOX-1 antibody reduced LOX-1 mRNA expression level in cells with oxLDL plus galectin-3 incubation. Compared to cells treated with oxLDL alone, reactive oxygen species (ROS) generation via nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and subsequent activation of p38 mitogen-activated protein kinases followed by nuclear factor kappa B (NF-κB) activation and related inflammatory responses including adhesion molecule expression, adhesiveness of monocytic cells, and IL-8 release were also aggravated in cells treated with galectin-3 combined with oxLDL. Compared to cells treated with galectin-3 plus oxLDL group. We found that LOX-1 antibody mitigated NADPH oxidase activity, p-38 up-regulation, NF-κB activation, and proinflammatory responses in cells treated with galectin-3 combined with oxLDL. We conclude that galectin-3 enhances endothelial LOX-1 expression and propose a new mechanism by which galectin-3 may promote endothelial dysfunction by inducing inflammation via LOX-1/ROS/p38/NF-κB-mediated signaling pathway., (© 2019 Wiley Periodicals, Inc.)

In China, cooking oil fume derived fine particulate matter (COF-derived PM 2.5 ) is a principal source of indoor air pollution. Here, we investigated cytotoxicity of COF-derived PM 2.5 , as well as the roles of VEGF, VEGFR2, MEK1/2, ERK1/2, and mTOR cascade in the inhibitory effects of COF-derived PM 2.5 , on angiogenesis in human umbilical vein endothelial cells (HUVECs). After exposure to COF-derived PM 2.5 , cell viability and tube formation, as well as protein and mRNA levels of VEGF, VEGFR2, MEK1/2, ERK1/2, and mTOR in HUVECs were measured. Cell viability and number of tubes reduced dose-dependently after COF-derived PM2.5 and SU5416 treatment. In addition, SU5416 and VEGF significantly affected tube formation. The protein and mRNA levels of VEGF, VEGFR2, MEK1/2, ERK1/2, and mTOR all tended to reduce with the increase of COF-derived PM2.5 concentrations. These findings demonstrate that VEGF, VEGFR2, MEK1/2, ERK1/2, and mTOR play key roles in COF-derived PM 2.5 induced inhibition of angiogenesis in HUVECs., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Magnesium (Mg) and its alloys as a type of different biodegradable materials have been used in the musculoskeletal field because of their excellent biocompatibility, biodegradability and mechanical properties similar to bone; besides, Mg could promote osteoblast differentiation in vitro and induce the formation of new bone in vivo. In the present study, we prepared the extracts of Mg-Zn-Mn alloy and examined their effects on the angiogenesis of human umbilical vein endothelial cells (HUVECs). In the present study, we prepared Mg-Zn-Mn alloy extracts of different concentrations and cultured HUVECs with these extracts. The DNA synthesis capacity, the cell viability, and the tube formation capacity of HUVECs could be significantly induced by 6.25% Mg alloy extract. In the meantime, the ratios of p-FGFR/FGFR, p-PI3K/PI3K, and p-AKT/AKT were significantly increased by 6.25% Mg alloy extract treatment, while decreased by FGFR/FGFR signaling pathway inhibitor BFJ398, indicating that 6.25% Mg alloy extract could promote the angiogenesis of HUVECs via activating FGF/FGFR signaling pathway. In conclusion, these data indicate that 6.25% Mg-Zn-Mn alloy extract induces the angiogenesis of HUVECs via FGF signaling pathway. Further in vivo experiments are needed to further confirm the present in vitro findings., (Copyright © 2019. Published by Elsevier Inc.)

The identification and removal of senescent cells is very important to improve human health and prolong life. In this study, we introduced a novel strategy of β-galactosidase (β-Gal) instructed peptide self-assembly to selectively form nanofibers and hydrogels in senescent cells. We demonstrated that the in situ formed nanofibers could alleviate endothelial cell senescence by reducing p53, p21, and p16 INK4a expression levels. We also demonstrated that our strategy could selectively remove senescent endothelial cells by inducing cell apoptosis, with an increase in the BAX/BCL-2 ratio and caspase-3 expression. Our study reports the first example of enzyme-instructed self-assembly (EISA) by a sugar hydrolase, which may lead to the development of supramolecular nanomaterials for the diagnosis and treatment of many diseases, such as cancer, and for other applications, such as wound healing and senescence.

Most achievements to engineer blood vessels are based on multiple-step manipulations such as manual sheet rolling or sequential cell seeding followed by scaffold degradation. Here, we propose a one-step strategy using a microfluidic coextrusion device to produce mature functional blood vessels. A hollow alginate hydrogel tube is internally coated with extracellular matrix to direct the self-assembly of a mixture of endothelial cells (ECs) and smooth muscle cells (SMCs). The resulting vascular structure has the correct configuration of lumen, an inner lining of ECs, and outer sheath of SMCs. These "vesseloids" reach homeostasis within a day and exhibit the following properties expected for functional vessels (i) quiescence, (ii) perfusability, and (iii) contractility in response to vasoconstrictor agents. Together, these findings provide an original and simple strategy to generate functional artificial vessels and pave the way for further developments in vascular graft and tissue engineering and for deciphering the angiogenesis process.

Angiogenesis is driven by the coordinated collective branching of specialized leading "tip" and trailing "stalk" endothelial cells (ECs). While Notch-regulated negative feedback suppresses excessive tip selection, roles for positive feedback in EC identity decisions remain unexplored. Here, by integrating computational modeling with in vivo experimentation, we reveal that positive feedback critically modulates the magnitude, timing, and robustness of angiogenic responses. In silico modeling predicts that positive-feedback-mediated amplification of VEGF signaling generates an ultrasensitive bistable switch that underpins quick and robust tip-stalk decisions. In agreement, we define a positive-feedback loop exhibiting these properties in vivo, whereby Vegf-induced expression of the atypical tetraspanin, tm4sf18, amplifies Vegf signaling to dictate the speed and robustness of EC selection for angiogenesis. Consequently, tm4sf18 mutant zebrafish select fewer motile ECs and exhibit stunted hypocellular vessels with unstable tip identity that is severely perturbed by even subtle Vegfr attenuation. Hence, positive feedback spatiotemporally shapes the angiogenic switch to ultimately modulate vascular network topology., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Angiogenesis is critical to cancer development and metastasis. However, anti-angiogenic agents have only had modest therapeutic success, partly due to an incomplete understanding of tumor endothelial cell (EC) biology. We previously reported that the microRNA (miR)-200 family inhibits metastasis through regulation of tumor angiogenesis, but the underlying molecular mechanisms are poorly characterized. Here, using integrated bioinformatics approaches, we identified the RNA-binding protein (RBP) quaking (QKI) as a leading miR-200b endothelial target with previously unappreciated roles in the tumor microenvironment in lung cancer. In lung cancer samples, both miR-200b suppression and QKI overexpression corresponded with tumor ECs relative to normal ECs, and QKI silencing phenocopied miR-200b-mediated inhibition of sprouting. Additionally, both cancer cell and endothelial QKI expression in patient samples significantly corresponded with poor survival and correlated with angiogenic indices. QKI supported EC function by stabilizing cyclin D1 (CCND1) mRNA to promote EC G1/S cell cycle transition and proliferation. Both nanoparticle-mediated RNA interference of endothelial QKI expression and palbociclib blockade of CCND1 function potently inhibited metastasis in concert with significant effects on tumor vasculature. Altogether, this work demonstrates the clinical relevance and therapeutic potential of a novel, actionable miR/RBP axis in tumor angiogenesis and metastasis.

Human aging is a physiological process characterized by a chronic low-grade inflammation. Senescence may affect endothelial cells, subsequently involved in the most common age-related diseases (ARDs), as well as mesenchymal stem cells (MSCs) with an impairment of their properties in tissues regeneration. Endothelial cells seem to be able to exert a paracrine effect on BM-MSCs through the secretion of pro-inflammatory factors. This work is aimed to evaluate if the senescent status of human umbilical vein endothelial cells (HUVECs) could affect bone marrow derived MSCs (BM-MSCs) proliferative ability and stemness. HUVECs were cultured until the senescence status. Young (passage 3) and senescent HUVECs (passage 13) were indirectly co-cultured with BM-MSCs for 8 days in order to evaluate the effect of their senescence status on proliferative ability and stemness of MSCs. The co-culture of senescent HUVECs with BM-MSCs was associated with a reduced proliferative ability of BM-MSCs, an enforced pro-inflammatory phenotype of BM-MSCs (increased synthesis of proinflammatory cytokines such as IL-6 and TNF-α) and an increased expression of miR-126a-3p, in association with a significant decrease of SOX2, a stemmness- associated gene, targeted by miR-126a-3p. A more general IPA analysis, revealed as miR-126a-3p also modulates the expression of IRS1, IRS2, IL6ST and PIK3R2, all targets that enforce the hypothesis that senescent endothelial cells may reduce the proliferative ability and the stemness phenotype of bone marrow-derived mesenchymal stem cells., (Copyright © 2019. Published by Elsevier Inc.)

Researches have been focusing on the role of Slit2 in angiogenesis, specifically in cell migration and vessel permeability. Nevertheless, the role of Slit2-N, the bioactive fragment of Slit2, in the proliferation of vascular endothelia in choroidal neovascularization and some related mechanisms have not been studied yet. Thus, our study aimed to explore the role of Slit2-N in proliferation of vascular endothelia and the related mechanisms in choroidal neovascularization. Fluorescein isothiocyanate perfusion and HE staining were performed to evaluate volumes of choroidal neovascularization lesions. The effect of Slit2-N on VEGF165-induced cell proliferation and some related mechanisms were detected by CCK8 assay, flow cytometry, siRNA transfection, and western blotting. We found that Slit2-N reduced volumes of laser-induced choroidal neovascularization networks in vivo. Results of the in vitro study showed Slit2-N reduced VEGF165-induced cell proliferation of both human umbilical vascular endothelial cells and human microvascular endothelial cells possibly via activation of AKT rather than that of ERK1/2. Additionally, Robo4, one of the receptors binding to Slit2-N, was involved in the inhibitory effect of Slit2-N. Generally, our findings revealed the inhibitory role of Slit2-N in proliferation of vascular endothelia and some related mechanisms, and presented some potential targets, molecules along Slit2-N-Robo4-AKT axis, to choroidal neovascularization therapy.

High glucose (HG) induces vascular injury in diabetes. Hydroxysafflor yellow A (HSYA) has been used to ameliorate ischemic cardiovascular diseases in China for many years. In the present study, we assessed whether HSYA has a potential protective role in HG-induced human umbilical vein endothelial cell (HUVEC) injury. Cell viability was determined with an 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Cell apoptosis was detected by fluorescein isothiocyanate/propidium iodide staining assay. The endothelial cell permeability was measured with a permeability assay. Cell adhesion molecule (CAM) expression, vascular endothelial growth factor, and basic fibroblast growth factor levels were detected with an enzyme-linked immunosorbent assay. Reactive oxygen species (ROS) formation was measured with a DCF-DA assay. Protein expression of NADPH oxidase 4 (NOX4) was measured by Western blotting. Our data indicated that HG increases HUVEC apoptosis, vascular permeability, monocyte adhesion, the level of CAMs, the formation of ROS, and NOX4 expression. Our data revealed that HG increases vascular injury, which is attenuated by HSYA. Because vascular inflammation has a key role in the development of diabetes mellitus, our results implied that HSYA is considered as a potential agent for diabetic vascular injury treatment.

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

Dental pulp has been revealed as an accessible and a rich source of mesenchymal stem cells (MSCs) and its biological potential is currently under intense investigation. MSCs from dental pulp stem cells (DPSCs) have been indicated as a heterogeneous population oriented not only in repairing dentine but also in maintaining vascular and nervous homeostasis of the teeth. We sought to verify the phenotype of cells isolated from dental pulp of young donors and to investigate in vitro their role as pericyte-like cells. Specifically, we evaluated how culture conditions can modulate expression of pericyte markers in DPSCs and their capacity to stabilize endothelial tubes in vitro. DPSCs cultured in standard conditions expressed MSC markers and demonstrated to contain a population expressing the pericyte marker NG2. These DPSCs were associated with low sprouting capacity in extra-cellular (EC) Matrix and limited ability in retaining tubes formed by endothelial cells in a coculture angiogenesis model. When cultured in endothelial growth medium (EGM)-2, DPSCs significantly upregulated NG2, and partially alpha-smooth muscle actin. The resulting population conserved the stem marker CD73, but was negative for calponin and endothelial markers. EGM-2-conditioned DPSCs showed a higher sprouting ability in EC Matrix and efficient association with human umbilical vein endothelial cells allowing the partial retention of endothelial tubes for several days. Among growth factors contained in EGM-2 we identified basic fibroblast growth factor (bFGF) as mainly responsible for NG2 upregulation and long-term stabilization of endothelial tubes. According to the in vitro analysis, DPSCs represent an effective source of pericytes and the appropriate culture conditions could result in a population with a promising ability to stabilize vessels and promote vascular maturation.

This work investigates the physicochemical properties and in vitro accuracy of a genetically engineered drug-delivery system based on elastin-like block recombinamers. The DNA recombinant techniques allowed us to create this smart complex polymer containing bioactive sequences for internalization, lysosome activation under acidic pH, and blockage of cellular growth by a small peptide inhibitor. The recombinant polymer reversibly self-assembled when the temperature was increased above 15 °C into nanoparticles with a diameter of 72 nm and negative surface charge. Furthermore, smart nanoparticles were shown to enter in the cells via clathrin-dependent endocytosis and properly blocked phosphorylation and consequent activation of Akt kinase. This system provoked apoptosis-mediated cell death in breast and colorectal cancer cells, which possess higher expression levels of Akt, whereas noncancerous cells, such as endothelial cells, fibroblasts, and mesenchymal stem cells, were not affected. Hence, we conclude that the conformational complexity of this smart elastin-like recombinamer leads to achieving successful drug delivery in targeted cells and could be a promising approach as nanocarriers with bioactive peptides to modulate multiple cellular processes involved in different diseases.

Testis-specific protein, Y-encoded like (TSPYL) family proteins (TSPYL1-6), which are members of the nucleosome assembly protein superfamily, have been determined to be involved in the regulation of various cellular functions. However, the potential role of TSPYL family proteins in endothelial cells (ECs) has not been determined. Here, we demonstrated that the expression of TSPYL5 is highly enriched in human ECs such as human umbilical vein endothelial cells (HUVECs) and human pluripotent stem cell-differentiated ECs (hPSC-ECs). Importantly, TSPYL5 overexpression was shown to promote EC proliferation and functions, such as migration and tube formation, by downregulating p53 expression. Adriamycin-induced senescence was markedly blocked by TSPYL5 overexpression. In addition, the TSPYL5 depletion-mediated loss of EC functions was blocked by p53 inhibition. Significantly, TSPYL5 overexpression promoted angiogenesis in Matrigel plug and wound repair in a mouse skin wound healing model in vivo. Our results suggest that TSPYL5, a novel angiogenic regulator, plays a key role in maintaining endothelial integrity and function. These findings extend the understanding of TSPYL5-dependent mechanisms underlying the regulation of p53-related functions in ECs.

Decellularized matrices offer a beneficial substitute for biomimetic scaffolds in tissue engineering. The current study examines the potential of decellularized placental vessel sleeves (PVS) as a periosteal protective sleeve to enhance bone regeneration in embryonic day 18 chick femurs contained within the PVS and cultured organotypically over a 10 day period. The femurs are inserted into decellularized biocompatibility-tested PVS and maintained in an organotypic culture for a period of 10 days. In femurs containing decellularized PVS, a significant increase in bone volume (p < 0.001) is evident, demonstrated by microcomputed tomography (µCT) compared to femurs without PVS. Histological and immunohistological analyses reveal extensive integration of decellularized PVS with the bone periosteum, and enhanced conservation of bone architecture within the PVS. In addition, the expressions of hypoxia inducible factor-1 alpha (HIF-1α), type II collagen (COL-II), and proteoglycans are observed, indicating a possible repair mechanism via a cartilaginous stage of the bone tissue within the sleeve. The use of decellularized matrices like PVS offers a promising therapeutic strategy in surgical tissue replacement, promoting biocompatibility and architecture of the tissue as well as a factor-rich niche environment with negligible immunogenicity., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Escin, a natural triterpene saponin mixture obtained from the horse chestnut tree (Aesculus hippocastanum), has been used for the treatment of chronic venous insufficiency (CVI), hemorrhoids, and edema. However, it is unclear how escin protects against endothelial barrier dysfunction induced by pro-inflammatory high mobility group protein 1 (HMGB1). Here, we report that escin can suppress (a) HMGB1-induced overexpression of the aquaporin-1 (AQP1) water channel in endothelial cells and (b) HMGB1-induced increases in endothelial cell permeability. This is the first report that escin inhibits AQP1 and alleviates barrier dysfunction in HMGB1-induced inflammatory response., (© 2019 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Hybrid small-diameter tubes were fabricated by wrapping decellularized aortic intima-media sheets around a tubular stainless steel mandrel with diameter 4 mm, and then by coating with electrospun segmented polyurethane. The synthetic coat was deposited uniformly to a thickness of about 0.5-3.5 μm depending on the duration of electrospinning. Resistance to luminal pressure, burst strength, and stiffness increased with the thickness of the electrospun coat, suggesting that the synthetic fabric reinforces the reconstructed acellular aortic intima-media. Human umbilical vein endothelial cells seeded on the inner surface acquired flagstone morphology, while normal human dermal fibroblasts seeded on the outer surface proliferated well and partly migrated into deeper layers. Collectively, the data suggest that reinforcing decellularized aortic intima-media with electrospun fibers generates a small-diameter hybrid blood vessel with good biocompatibility and suitable mechanical properties. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1064-1070, 2019., (© 2019 Wiley Periodicals, Inc.)

Concentration gradients of biochemical stimuli such as morphogens play a critical role in directing cell fate patterning across species and throughout development but are not commonly recapitulated in vitro. While in vitro biomolecule gradients have been generated using customized microfluidic platforms, broad implementation has been limited because these platforms introduce new variables to cell culture such as externally driven flow, culture in a specialized matrix, or extended time for in situ long range diffusion. Here we introduce a method that enables preforming and then transferring user-controlled gradients to cells in standard "open" cultures. Our gradient patterning devices are modular and decoupled from the culture substrate. We find that gradient generation and transfer are predictable by finite element modeling and that device and loading parameters can be used to tune the stimulus pattern. Furthermore, we demonstrate use of these devices to spatially define morphogen signal gradients and direct peri-gastrulation fate stratification of human pluripotent stem cells. This method for extrinsic application of biochemical signal gradients can thus be used to spatially influence cellular fate decisions in a user-controlled manner.

Leishmaniasis is one of the most important parasitic diseases after malaria. The standard treatment of leishmaniasis includes pentavalent antimonials (SbV); however, these drugs are associated with serious adverse effects. There have been very few studies pertaining to their side effects and mechanism of action in the fetus. This investigation examines the effects of meglumine antimoniate (MA) on the survival rate, angiogenesis and cellular apoptosis in the human umbilical vein endothelial cells (HUVECs). HUVECs were treated with varying doses of MA (100-800 μg/ml) for 24, 48 and 72 h and the survival rate was studied by colorimetric assay, flow cytometry, immunocytochemistry, migration (scratch) assay and tube formation assay. The results of quantitative real-time PCR (qPCR) studies indicated that the most important genes involved in presenting angiogenesis included VEGF and its receptors (Kdr and Flt-1), NP1 and Hif-1α genes including the anti-apoptotic gene of Bcl2, were significantly reduced compared to the control group (p < 0.05). In contrast, the most leading genes involved in the phenomenon of apoptosis were P53, Bax, Bak, Apaf-1 and caspases 3, 8 and 9, which were significantly up regulated compared to the control group (p < 0.05)., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Aim: The aim of this study was to evaluate the antihypertensive effect of Xin Mai Jia (XMJ) and to explore the mechanism of its hypotensive effect., Methods: A total of 50 spontaneously hypertensive rats (SHR) were randomised into five groups. A total of 30 Wistar-Kyoto rats were randomised into three groups, comprising the control group. All of the rats were administered medicine through a gastrogavage once a day for 8 weeks. The tail-cuff method was applied to their monitor blood pressure. After 8 weeks of treatment, serum NO, SOD activity, MDA level, ET, ALD, AngII, RE, and CGRP in the serum were detected in all of the rats. Pathological changes in the aorta were observed via haematoxylin-eosin (HE) and immunohistochemical staining. Vasodilation function was assessed by measuring acetylcholine-induced vessel relaxation in the rats' organ chambers. The function of the mesenteric arteries was measured using DMT wire myography. Human aortic smooth muscle cells (HASMCs) and human umbilical vein endothelial cells (HUVECs) injury models were induced by hydrogen peroxide (H 2 O 2 ). HASMCs and HUVECs were injured by H 2 O 2 and then exposed to various drugs. HASMC and HUVEC migration was evaluated using the cell scratch test. The expression of the AT1 receptors (AT1R) in the HASMCs was detected via immunofluorescence (IFC) assay., Results: After 8 weeks of treatment, XMJ reduced the systolic blood pressure of the SHR. XMJ significantly reduced the serum RE, AngII, ALD, and ET-1 levels and increased the content of CGRP and NO in the SHR, upregulated the SOD content, and downregulated MDA level of the SHR. XMJ improved pathological damage of the aorta to varying degrees, decreased the expression of AT1R in the SHR aortic vessels, and improved the mesenteric microvascular relaxation of the SHR. Cell experiments confirmed that XMJ inhibited the migration of the HUVECs and HASMCs induced by H 2 O 2 and the expression of AT1R in the HASMCs., Conclusion: XMJ had satisfactory hypotensive action on the SHR in this study. Its mechanism may be associated with inhibiting RAAS activity and improving RAAS function, inhibiting hypertensive-induced vascular diastolic dysfunction, and improving vascular endothelial function., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Objectives: We aimed to accelerate angiogenesis in pulp regeneration by modulating ephrinB2 expression in stem cells from apical papilla (SCAPs)., Materials and Methods: Stem cells from apical papilla were transducted with ephrinB2-lentiviral expression vector (ephrinB2-SCAPs) in experimental group and green fluorescent protein (GFP-SCAPs) in control group. The transduction efficiency was confirmed by real-time PCR and Western blot assays. MTT assay was performed to detect the proliferative capacity of SCAPs after transduction. In vitro Matrigel assay and in vivo Matrigel plug assay were carried out to evaluate the angiogenic capacity., Results: Results showed that ephrinB2-SCAPs had significantly higher ephrinB2 expression than GFP-SCAPs. EphrinB2-SCAPs upregulated vascular endothelial growth factor (VEGF) secretion under hypoxia. In vitro Matrigel assay demonstrated that human umbilical vein endothelial cells (HUVECs) cocultured with ephrinB2-SCAPs under hypoxia formed vascular-like structures earlier than GFP-SCAPs. Animal experiments confirmed that SCAPs co-transplanted with HUVECs enabled to generate greater amount of blood vessels than SCAPs alone. EphrinB2-SCAPs produced increased number of blood vessels with references to GFP-SCAPs, and those co-transplanted with HUVECs generated vessels with larger and functional tubule volumes., Conclusions: Regulating ephrinB2 expression in SCAPs may act as a new avenue for enhancing angiogenesis in dental pulp regeneration., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.)

Ethnopharmacological Relevance: Hepatocellular carcinoma (HCC) is among the most common malignancies. Signal transducer and activator of transcription 3 (STAT3), with abnormal expression and constitutive activation, has been reported to promote proliferation, metastasis, survival and angiogenesis of HCC cells. Rheum palmatum (RP), a traditional Chinese medicinal herb, exhibited tumor-suppressing effects in multiple human cancers, but its potential functions in HCC remain unexplored., Aim of the Study: This study aimed to examine the involvement of STAT3 signaling in the anti-HCC effects of RP extract., Materials and Methods: SMMC-7721 and HepG2 HCC cell lines were treated with RP extract for 24 h, and then viability, migration, and invasion of HCC cells and angiogenesis of human umbilical vein endothelial cells (HUVECs) were analyzed using MTS, wound-healing, Transwell invasion and tube formation assays, respectively. Western blotting and immunohistochemistry (IHC) were used to examine the activation of key molecules in STAT3 signaling, including STAT3, JAK2, and Src. Additionally, we explored the in vivo antitumor effects of RP extract in a xenograft tumor nude mouse model of HCC., Results: The result showed that RP extract reduced viability, migration, and invasion of SMMC-7721 and HepG2 cells and angiogenesis of HUVECs. It suppressed the phosphorylation of STAT3 and its upstream kinases including JAK2 and Src. In addition, RP extract treatment downregulated STAT3 target genes, including survivin, Bcl-xL, Mcl-1, Bcl-2, MMP-2, MMP-9, Cyclin D1, CDK4, c-Myc, and VEGF-C. Furthermore, RP extract suppressed the xenograft tumor growth and activation of STAT3 in xenograft tumor mice., Conclusion: Collectively, the results showed that RP extract prevented HCC progression by inhibiting STAT3, and might be useful for the treatment of HCC., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Angiogenesis plays a critical role in embryo development, tissue repair, tumor growth and wound healing. In the present study, we investigated the role of the serine/threonine kinase Akt in angiogenesis. Silencing of Akt1 in human umbilical vein endothelial cells significantly inhibited vascular endothelial growth factor (VEGF)-induced capillary-like tube formation. Mice lacking Akt1 exhibited impaired retinal angiogenesis with delayed endothelial cell (EC) proliferation. In addition, VEGF-induced corneal angiogenesis and tumor development were significantly inhibited in mice lacking Akt1. Loss of Akt1 resulted in reduced angiogenic sprouting, as well as the proliferation of ECs and mural cells. Addition of culture supernatant of vascular smooth muscle cells (VSMCs) in which Akt1 was silenced suppressed tube formation, the stability of preformed tubes and the proliferation of ECs. In addition, attachment of VSMCs to ECs was significantly reduced in cells in which Akt1 was silenced. Mural cell coverage of retinal vasculature was reduced in mice lacking Akt1. Finally, mice lacking Akt1 showed severe retinal hemorrhage compared to the wild-type. These results suggest that the regulation of EC function and mural cell coverage by Akt1 is important for blood vessel maturation during angiogenesis., Competing Interests: The authors declare no conflict of interest.

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

Rationale: Endothelial barrier function depends on the proper localization and function of the adherens junction protein VE (vascular endothelial)-cadherin. Previous studies have suggested a functional relationship between integrin-mediated adhesion complexes and VE-cadherin yet the underlying molecular links are unclear. Binding of the cytoskeletal adaptor protein talin to the β-integrin cytoplasmic domain is a key final step in regulating the affinity of integrins for extracellular ligands (activation) but the role of integrin activation in VE-cadherin mediated endothelial barrier function is unknown., Objective: To test the requirement of talin-dependent activation of β1 integrin in VE-cadherin organization and endothelial cell (EC) barrier function., Methods and Results: EC-specific deletion of talin in adult mice resulted in impaired stability of intestinal microvascular blood vessels, hemorrhage, and death. Talin-deficient endothelium showed altered VE-cadherin organization at EC junctions in vivo. shRNA (short hairpin RNA)-mediated knockdown of talin1 expression in cultured ECs led to increased radial actin stress fibers, increased adherens junction width and increased endothelial monolayer permeability measured by electrical cell-substrate impedance sensing. Restoring β1-integrin activation in talin-deficient cells with a β1-integrin activating antibody normalized both VE-cadherin organization and EC barrier function. In addition, VE-cadherin organization was normalized by reexpression of talin or integrin activating talin head domain but not a talin head domain mutant that is selectively deficient in activating integrins., Conclusions: Talin-dependent activation of EC β1-integrin stabilizes VE-cadherin at endothelial junctions and promotes endothelial barrier function.

Background: Quercetin, a flavonoid antioxidant, has been found to exert therapeutic effects in diabetic condition. Autophagy represents a homeostatic cellular mechanism for the turnover of unfolds proteins and damaged organelles through a lysosome-dependent degradation manner. We speculated that quercetin could protect endothelial cells against high glucose-induced damage by promoting autophagic responses., Methods: HUVECs viability was evaluated by MTT method. Griess and TBARS assays were used to monitor the levels of NO and MDA, respectively. Intracellular ROS generation was determined in DCFDA-stained cells analyzed by flow cytometry. To investigate the role of quercetin in endothelial cell migratory behavior, we used a scratch test. The level of autophagy proteins LC3, Beclin-1 and P62 were measured by western blotting technique., Results: Our results showed that quercetin had the potential to increase cell survival after exposure to high glucose (P < 0.05). Total levels of oxidative stress markers were profoundly decreased and the activity of GSH was increased by quercetin (P < 0.05). High glucose suppressed HUVECs migration to the scratched area (P < 0.05). However, a significant stimulation in cell migration was observed after exposure to quercetin (P < 0.05). Based on data, autophagy was blocked at the late stage by high glucose concentration while quercetin enhanced autophagic response by reducing the P62 level coincided with the induction of Beclin-1 and LC3-II to LC3-I ratio (P < 0.05). All these beneficial effects were reversed by 3-methyladenine as an autophagy inhibitor., Conclusion: Together, our data suggest that quercetin could protect HUVECs from high glucose induced-damage possibly by activation of the autophagy response., (Copyright © 2018. Published by Elsevier GmbH.)

Despite advances in the development of materials for cardiovascular devices, current strategies generally lack the thromboresistance of the native endothelium both in terms of efficacy and longevity. To harness this innate hemostatic regulation and improve long-term hemocompatibility, biohybrid devices are designed to promote endothelialization. Much of the research effort to date has focused on the use of extracellular matrix (ECM)-mimics and coatings to promote endothelial cell adhesion and migration with less attention given to the effect of the supported ECM binding events on hemostatic regulation. In this study, we developed integrin-targeted hydrogels to investigate the individual and combined effects of integrin binding events supported by many ECM-based coatings (α1β1, α2β1, α5β1, αvβ3). Targeted endothelial cell integrin interactions were first confirmed with antibody blocking studies and then correlated with gene expression of hemostatic regulators and a functional assay of platelet attachment and activation. Surfaces that targeted integrins α1β1 and α2β1 resulted in an endothelial cell layer that exhibited a thromboresistant phenotype with an associated reduction in platelet attachment and activation. It is anticipated that identification of specific integrins that promote endothelial cell adhesion as well as thromboresistance will enable the design of cardiovascular materials with improved long-term hemocompatibility.

Formononetin is one of the main active components of traditional Chinese medicine red clover (Trifolium pratense L). Prior studies have demonstrated that formononetin is a typical phytoestrogen and exhibits an estrogen-like effect that protects the cardiovascular system with minimal side effects. In this study, we further investigated the role of formononetin in human umbilical vein endothelial cells (HUVECs) and its potential molecular mechanisms. We founded that formononetin promotes proliferation and migration of HUVECs as assessed by the MTT and wound healing assays. Meanwhile, insulin-like growth factor 1 (IGF-1) and intercellular adhesion molecule 1 (ICAM-1) protein levels were increased in a dose-dependent manner. Our findings illustrated that formononetin exhibits a protective effect on HUVECs, and the molecular mechanisms may correlate with IGF-1R and ICAM-1, as well as upregulation of vascular endothelial growth factor (VEGF) and activation of extracellular signal-regulated kinase (ERK)., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Recent advances in developing in vitro tissue models show that function of hepatocytes is altered in when cultured in 3D configuration and co-culturing with various non-parenchymal cells. However, tissue source for such non-parenchymal cells on viability and metabolic products of hepatocytes have not been explored. In this study, we evaluated the effect of 2D and 3D cultures either with HepaRG cells alone or in combination with liver sinusoidal endothelial cells (LSECs) and human umbilical vein ECs (HUVECs). For 3D cultures, we used chitosan-gelatin porous structures formed by freeze-drying. We cultured cells for 8 days before challenging with 1 mm acetaminophen (APAP) and assessed APAP, APAP-sulfate and APAP-glucuronide for 24 hours at 6-hour time intervals using high-performance liquid chromatography. We used multiple methods (phase contrast, confocal and scanning electron microscopy and histology via hematoxylin and eosin staining) to ensure cell distribution. We also measured total protein content and albumin secretion and viability. HUVEC 3D co-cultures showed the lowest HepaRG cell viability, while both 2D and 3D LSEC co-cultures had highest HepaRG cell viability. In addition, 3D cultures had significantly higher EC viability relative to 2D cultures. Further, HUVEC co-cultures showed reduced total protein content and albumin expression as early as day 4. However, urea production on a total protein content basis did not change. In addition, LSEC 3D co-cultures had the highest APAP conversion with reduced APAP-sulfate and APAP-glucuronide formation. CYP3A4 was higher in co-culture with HUVEC for 2D and 3D cultures. In conclusion, HepaRG cells with EC co-cultures demonstrated sensitivity to the EC line used., (© 2018 John Wiley & Sons, Ltd.)

Purpose: Peristaltic pumps (PP) are favored in flow bioreactors for their non-contact sterile design. But they produce pulsatile flow, which is consequential for the cultured cells. A novel pulse damper (PD) is reported for pulsatility elimination., Methods: The PD design was implemented to target static pressure pulsatility and flow rate (velocity) pulsatility from a PP. Damping effectiveness was tested in a macro-scale, closed-loop recirculating bioreactor mimicking the aortic arch at flow rates up to (4 L/min). Time-resolved particle image velocimetry was used to characterize the velocity field. Endothelial cells (EC) were grown in the bioreactor, and subjected to continuous flow for 15 min with or without PD., Results: The PD was found to be nearly 90% effective at reducing pulsatility. The EC exposed to low PP flow without PD exhibited distress signaling in the form of increased ERK1/2 phosphorylation (2.5 folds) when compared to those exposed to the same flow with PD. At high pump flow without PD, the cells detached and did not survive, while they were perfectly healthy with PD., Conclusions: Flow pulsatility from PP causes EC distress at low flow and cell detachment at high flow. Elevated temporal shear stress gradient combined with elevated shear stress magnitude at high flow are believed to be the cause of cell detachment and death. The proposed PD design was effective at minimizing the hemodynamic stressors in the pump's output, demonstrably reducing cell distress. Adoption of the proposed PD design in flow bioreactors should improve experimental protocols.

The involvement of intercellular interactions in various biological events indicates the importance of studying cell-cell interactions using fluid model surfaces. Here, we propose a fluid surface composed of a self-assembled monolayer (SAM) and poly(ethylene glycol)-conjugated phospholipid (PEG-lipid) derivatives, which can be an alternative to supported lipid membranes. The modification of SAM surfaces with PEG-lipids carrying functional peptides resulted in the formation of the fluid surfaces with different mobility, which was quantitatively determined by quartz crystal microbalance with dissipation (QCM-D) and fluorescence recovery after photobleaching (FRAP). Different types of fluid surfaces with calculated diffusion coefficients between 0.9 ± 0.25 and 0.16 ± 0.03 μm 2 /sec for PEG-lipids derivatives were fabricated, onto which arginylglycylaspartate (RGD) peptides were immobilized for cell adhesion, and compared to solid surfaces with the same surface density of RGD peptides. The fluid surfaces revealed that cell adhesions of epithelial cells (MCF-10 A) and human umbilical vein endothelial cells (HUVEC) could not be established on the surfaces with higher fluidity, while cells could adhere onto surfaces with lower fluidity, where the lateral diffusion of PEG-lipids was approximately 20 times lower, and solid surfaces. Interestingly, cells that adhered onto the surface with lower fluidity proliferated at a normal rate while maintaining their round morphology, which was a different shape from that observed on solid surfaces. Thus, the scaffold fluidity greatly influenced cell adhesion behaviors, demonstrating that it is an important parameter for designing novel biomimetic scaffolds for biomedical applications., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Objective: Pyroptosis is an inflammatory form of programmed cell death. This phenomenon has been recently reported to play an important role in radiation-induced normal tissue injury. Connexin43 (Cx43) is a gap junction protein that regulates cell growth and apoptosis. In this study, we investigated the effect of Cx43 on X-ray-induced pyroptosis in the human umbilical vein endothelial cells (HUVECs)., Methods: HUVECs, Cx43 overexpression, and Cx43 knockdown strains were irradiated with 10 Gy. Proteins were detected using western blot analysis. Cell pyroptosis was evaluated using the fluorescence-labeled inhibitor of caspase assay (FLICA) and propidium iodide staining through flow cytometry and confocal microscopy. Cell morphology and cytotoxicity were detected by scanning electron microscopy and lactate dehydrogenase release assay, respectively., Results: Irradiation with 10 Gy X-ray induced pyroptosis in the HUVECs and reduced Cx43 expression. The pyroptosis in the HUVECs was significantly attenuated by overexpression of Cx43 as it decreased the level of active caspase-1. However, interference of Cx43 expression with siRNA significantly promoted pyroptosis by increasing the active caspase-1 level. Pannexin1 (Panx1), a gap junction protein regulates pyroptosis, and its cleaved form is used to evaluate channel opening and active state. The level of cleaved Panx1 in the HUVECs and Cx43 knockdown strains increased in the presence of X-ray, but decreased in the Cx43 overexpression strains. Furthermore, interference of Panx1 with siRNA alleviated the upregulation of pyroptosis caused by Cx43 knockdown., Conclusion: Results suggest that single high-dose X-ray irradiation induces pyroptosis in the HUVECs. In addition, Cx43 regulates pyroptosis directly by activating caspase-1 or indirectly by cleaving Panx1., (Copyright © 2019 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

The current study used an ex vivo [embryonic day (E)18] chick femur defect model to examine the bone regenerative capacity of implanted 3-dimensional (3D) skeletal-endothelial cell constructs. Human bone marrow stromal cell (HBMSC) and HUVEC spheroids were implanted within a bone defect site to determine the osteogenic potential of the skeletal-endothelial cell unit. Cells were pelleted as co- or monocell spheroids and placed within 1-mm-drill defects in the mid-diaphysis of E18 chick femurs and cultured organotypically for 10 d. Micro-computed tomography analysis revealed significantly ( P = 0.0001) increased levels of bone volume (BV) and BV/tissue volume ratio in all cell-pellet groups compared with the sham defect group. The highest increase was seen in BV in femurs containing the HUVEC and HBMSC monocell constructs. Type II collagen expression was particularly pronounced within the cell spheres containing HBMSCs and HUVECs, and CD31-positive cell clusters were prominent within HUVEC-implanted defects. These studies demonstrate the importance of the 3D osteogenic-endothelial niche interaction in bone regeneration. Elucidating the component cell interactions in the osteogenic-vascular niche and the role of exogenous factors in driving these osteogenic processes will aid the development of better bone reparative strategies.-Inglis, S., Kanczler, J. M., Oreffo, R. O. C. 3D human bone marrow stromal and endothelial cell spheres promote bone healing in an osteogenic niche.

Fuchs endothelial dystrophy (FED) and late cornea allograft failure of cornea transplantation are associated with human corneal endothelial cells (HCEC) senescence. Kojic acid has various functions, however, its anti-senescence effect has never been identified. In this study, we investigated the anti-senescence effect of kojic acid on HCEC. Cell viability, migration ability and senescence were evaluated by MTT assay, migration assay, and senescence-associated beta-galactosidase (SA-β-Gal) staining, respectively. Senescence-related protein expression was analyzed by western blotting and immunofluorescence assay. Angiogenesis of human umbilical vein endothelial cells (HUVEC) was examined by tube formation assay and spheroid sprouting assay. The results showed that kojic acid could inhibit HCEC senescence, characterized by enhancing migration, decreasing the levels of SA-β-Gal staining, galectin 8, laminin α1, laminin α2, laminin γ1 and p21, and increasing that of p-NF-κB of senescent HCEC. The p-NF-κB inhibitor could reverse the anti-senescent effect of kojic acid, and p21 siRNA showed similar anti-senescence effect with kojic acid. In addition, kojic acid could alleviate HUVEC tube formation induced by senescent HCEC, which could be reversed by p-NF-κB inhibitor. The p21 siRNA could alleviate HUVEC spheroid sprouting induced by senescent HCEC. These results indicated that kojic acid might inhibit HCEC senescence and following resulted angiogenesis via NF-κB and p21 signaling pathways, possibly through downregulation of galectin 8 and laminins. Therefore, kojic acid is a promising drug for HCEC senescence-related diseases such as FED and late cornea allograft failure., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

During chronic inflammation, neutrophils acting locally as effector cells not only activate antibacterial defense but also promote the inflammatory response. Interleukin 8 (IL-8), the main cytokine produced by activated neutrophils, positively correlates with the severity of respiratory tract diseases. By screening European plants traditionally used for treating respiratory tract diseases, we found that extracts of aerial parts of Eupatorium cannabinum inhibit IL-8 release from neutrophils. Using bioassay-guided fractionation, we identified five sesquiterpene lactones, eupatoriopicrin (1), 5'-deoxyeupatoriopicrin (2), hiyodorilactone A (3), 3-hydroxy-5'- O-acetyleupatoriopicrin = hiyodorilactone D (4), and hiyodorilactone B (5), that efficiently (IC 50 < 1 μM) inhibited IL-8 and TNF-α release in lipopolysaccharide (LPS)-stimulated human neutrophils. Moreover, all these sesquiterpene lactones suppressed the adhesion of human neutrophils to an endothelial monolayer by downregulating the expression of the β2 integrin CD11b/CD18 on the neutrophil surface. Furthermore, eupatoriopicrin efficiently suppressed LPS-induced phosphorylation of p38 MAPK and ERK and attenuated neutrophil infiltration in the thioglycolate-induced peritonitis model in mice. Altogether, these results demonstrate the potential of the sesquiterpene lactone eupatoriopicrin as a lead substance for targeting inflammation.

The development of new capillary networks in engineered constructs is essential for their survival and their integration with the host tissue. It has recently been demonstrated that ELR-based hydrogels encoding different bioactivities are able to modulate their interaction with the host after injection or implantation, as indicated by an increase in cell adhesion and the ability to trigger vascularization processes. Accordingly, the aim of this study was to increase their angiogenic ability both in vitro and in vivo using a small VEGF mimetic peptide named QK, which was tethered chemically to ELR-based hydrogels containing cell-adhesion sequences in their backbone, such as REDV and RGD, as well as a proteolytic site (VGVAPG). In vitro studies were performed using a co-culture of endothelial and fibroblast cells encapsulated into the ELR-based hydrogels in order to determine cell proliferation after 21 days of culture, as well as the number of cell-cell interactions. It was found that although the presence of this peptide does not influence the morphological and rheological properties of these hydrogels, it has an effect on cell behaviour, inducing an increase in cell proliferation and the formation of endothelial cell clusters. In vivo studies demonstrate that the QK peptide enhances the formation of prominent functional capillaries at three weeks post-injection, as confirmed by H&E staining and CD31 immunohistochemistry. The newly formed functional microvasculature ensures perfusion and connection with surrounding tissues. These results show that ELR-QK hydrogels increase capillary network formation and are therefore attractive candidates for application in tissue regeneration, for example for the treatment of cardiovascular diseases such as myocardial infarction or ischemia.

Exchange protein activated by cyclic AMP (EPAC1) suppresses multiple inflammatory actions in vascular endothelial cells (VECs), partly due to its ability to induce expression of the suppressor of cytokine signalling 3 (SOCS3) gene, the protein product of which inhibits interleukin 6 (IL6) signalling through the JAK/STAT3 pathway. Here, for the first time, we use the non-cyclic nucleotide EPAC1 agonist, I942, to determine its actions on cellular EPAC1 activity and cyclic AMP-regulated gene expression in VECs. We demonstrate that I942 promotes EPAC1 and Rap1 activation in HEK293T cells and induces SOCS3 expression and suppresses IL6-stimulated JAK/STAT3 signalling in HUVECs. SOCS3 induction by I942 in HUVECs was blocked by the EPAC1 antagonist, ESI-09, and EPAC1 siRNA, but not by the broad-spectrum protein kinase A (PKA) inhibitor, H89, indicating that I942 regulates SOCS3 gene expression through EPAC1. RNA sequencing was carried out to further identify I942-regulated genes in HUVECs. This identified 425 I942-regulated genes that were also regulated by the EPAC1-selective cyclic AMP analogue, 007, and the cyclic AMP-elevating agents, forskolin and rolipram (F/R). The majority of genes identified were suppressed by I942, 007 and F/R treatment and many were involved in the control of key vascular functions, including the gene for the cell adhesion molecule, VCAM1. I942 and 007 also inhibited IL6-induced expression of VCAM1 at the protein level and blocked VCAM1-dependent monocyte adhesion to HUVECs. Overall, I942 represents the first non-cyclic nucleotide EPAC1 agonist in cells with the ability to suppress IL6 signalling and inflammatory gene expression in VECs., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

In this study, we prepared fractions containing multifunctional cationic peptides by separating the commercial soybean protein hydrolysate Hinute-AM into 20 fractions. These fractions contained peptides with various isoelectric points (pI), as indicated by ampholyte-free isoelectric focusing (autofocusing). Thus, we purified and identified the cationic peptides from fractions 19 and 20, which had pH values greater than 10, using reversed-phase high-performance liquid chromatography and matrix-assisted laser/desorption ionization-time-of-flight mass spectroscopy. Among 19 identified cationic peptides, NKNAKPPSPR, PGKKNAIV, KSGPGMSPR, NVSKPPRVV, RKVGAGGRKPLG, and LPCVIGGVPKRV had high pI values and were included as chemically synthesized peptides in assays of various functions, including lipopolysaccharide (LPS)-neutralizing and angiogenic activities. Chromogenic LPS-neutralizing assays using Limulus amebocyte lysates showed that 50% effective concentrations of these six peptides were between 1.63 and 2.65 μM, and were higher than that (0.12 μM) of polymyxin B. Moreover, in tube-formation assays in human umbilical vein endothelial cells, all of the six cationic peptides except LPCVIGGVPKRV exhibited angiogenic activities similar to those of the positive control LL-37. In addition, the six identified cationic peptides had no hemolytic activity at concentrations up to 500 μM in mammalian red blood cells. Our results demonstrate that five of the identified cationic peptides, excluding LPCVIGGVPKRV, have multiple functions and little or no hemolytic activity. These data indicate that fractions containing cationic peptides from Hinute-AM have the potential to be used as dietary supplements and functional ingredients in food products., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Cells interplay with their environment through mechanical and chemical interactions. To characterize this interplay, endothelial cells were cultured on polyacrylamide hydrogels of varying stiffness, coated with either fibronectin or collagen. We developed a novel analysis technique, complementary to traction force microscopy, to characterize the spatiotemporal evolution of cellular tractions: We identified subpopulations of tractions, termed traction foci, and tracked their magnitude and lifetime. Each focus consists of tractions associated with a local single peak of maximal traction. Individual foci were spread over a larger area in cells cultured on collagen relative to those on fibronectin and exerted higher tractions on stiffer hydrogels. We found that the trends with which forces increased with increasing hydrogel stiffness were different for foci and whole-cell measurements. These differences were explained by the number of foci and their average strength. While on fibronectin multiple short-lived weak foci contributed up to 30% to the total traction on hydrogels with intermediate stiffness, short-lived foci in such a number were not observed on collagen despite the higher tractions. Our approach allows for the use of existing traction force microscopy data to gain insight at the subcellular scale without molecular probes or spatial constraining of cellular tractions.

Forming functional blood vessel networks is a major clinical challenge in the fields of tissue engineering and therapeutic angiogenesis. Cell-based strategies to promote neovascularization have been widely explored, but cell sourcing remains a significant limitation. Induced-pluripotent stem cell-derived endothelial cells (iPSC-ECs) are a promising, potentially autologous, alternative cell source. However, it is unclear whether iPSC-ECs form the same robust microvasculature in vivo documented for other EC sources. In this study, we utilized a well-established in vivo model, in which ECs (iPSC-EC or human umbilical vein endothelial cells [HUVEC]) were coinjected with normal human lung fibroblasts (NHLFs) and a fibrin matrix into the dorsal flank of severe combined immunodeficiency mice to assess their ability to form functional microvasculature. Qualitatively, iPSC-ECs were capable of vessel formation and perfusion and demonstrated similar vessel morphologies to HUVECs. However, quantitatively, iPSC-ECs exhibited a two-fold reduction in vessel density and a three-fold reduction in the number of perfused vessels compared with HUVECs. Further analysis revealed the presence of collagen-IV and α-smooth muscle actin were significantly lower around iPSC-EC/NHLF vasculature than in HUVEC/NHLF implants, suggesting reduced vessel maturity. Collectively, these results demonstrate the need for increased iPSC-EC maturation for clinical translation to be realized., (© 2018 Wiley Periodicals, Inc.)

The adhesion of monocytes to endothelial cells, which is mediated by adhesion molecules, plays a crucial role in the onset of atherosclerosis. Conjugated equine estrogen, which is widely used for estrogen-replacement therapy, contains both estrone sulfate and various nonhuman estrogens, including equilin. To investigate the association between various estrogen types and atherosclerosis risk, we examined their effect on adhesion-molecule expression in human umbilical vein endothelial cells (HUVECs). In estrogen-treated HUVECs, the mRNA and protein expression levels of adhesion molecules were quantified by real-time polymerase chain reaction and enzyme immunoassay. Additionally, a flow-chamber system was used to assess the effects of estrogens on the adherence of U937 monocytoid cells to HUVECs. Equilin, but not 17β-estradiol (E2) or other types of estrogen, significantly increased the mRNA (P < 0.01) and protein (P < 0.05) expression of the adhesion molecules E-selectin and intercellular adhesion molecule-1 as compared with levels in controls. Equilin treatment increased the adherence of U937 monocytoid cells to HUVECs relative to the that in the control (P < 0.05), decreased estrogen receptor (ER)β expression, and increased the expression of proteins involved in nuclear factor kappa-B (NF-κB) activation relative to levels in controls. Furthermore, the accumulation of NF-κB subunit p65 in HUVEC nuclei was promoted by equilin treatment. By contrast, E2 treatment neither increased the number of adhered monocytoid cells to HUVECs nor altered the expression of ERβ or NF-κB-activating proteins. Our findings suggest that in terms of the adhesion of monocytes at the onset of atherosclerosis, E2 may be preferable for estrogen-replacement therapy. Further studies comparing equilin treatment with that of E2 are needed to investigate their differential impacts on atherosclerosis., Competing Interests: The authors have declared that no competing interests exist.

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

Angiopoietin-1 (Ang-1) is a ligand of Tie-2 receptors that promotes survival, migration, and differentiation of endothelial cells (ECs). Recent studies have identified several microRNA (miRNA) families that either promote or inhibit angiogenesis. To date, the nature and functional importance of miRNAs in Ang-1-induced angiogenesis are unknown. Microarray screening of known miRNAs in human umbilical vein endothelial cells (HUVECs) revealed that the expressions of miR-103b, miR-330-5p, miR-557, miR-575, miR-1287-5p, and miR-1468-5p significantly decrease following exposure to Ang-1 for 24 h. Exposure to the angiogenesis factors angiopoietin-2 (Ang-2), vascular endothelial growth factor, fibroblast growth factor 2, and transforming growth factor β also inhibits miR-103b expression, but exerts varying effects on the other miRNAs. By overexpressing miR-103b, miR-330-5p, miR-557, miR-575, miR-1287-5p, and miR-1468-5p with selective mimics, we demonstrated that the pro-survival effects of Ang-1 are eliminated, Caspase-3 activity increases, and cell migration, proliferation, and capillary-like tube formation decreases. Conversely, transfection with selective miRNA inhibitors increases cell survival, inhibits Caspase-3 activity, and stimulates migration, proliferation and tube formation. miRNet miRNA-target gene network analyses revealed that miR-103, miR-330-5p, miR-557, miR-575, miR-1287-5p, and miR-1468-5p directly interact with 47, 95, 165, 108, 49, and 16 gene targets, respectively. Since many of these genes are positive regulators of angiogenic processes, we conclude that these miRNAs function as anti-angiogenic miRNAs and that their downregulation may be essential for Ang-1-induced angiogenesis to occur., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A perfect microenvironment facilitates the activated circulating tumor cells (CTCs) to spark the adhesion-invasion-extravasation metastatic cascade in their premetastatic niche. Platelet-CTC interaction contributes to the progression of tumor malignancy by protecting CTCs from shear stress and immunological assault, aiding CTCs entrapment in the capillary bed, enabling CTCs to successfully exit the bloodstream and enter the tissue, inducing epithelial-mesenchymal-like transition (EMT), and assisting in the establishment of metastatic foci. To prevent the cascade from sparking, we show that, the multifunctional S-nitrosocaptopril (CapNO) acts on both CTCs and platelets to interrupt platelet/CTCs interplay and adhesion to endothelium, thus inhibiting CTC-based pulmonary metastasis in vivo. The activated platelets cloak cancer HT29 cells, resulting in HT29-exhibiting platelet biomarkers CD61 and P-selectin positive. CapNO inhibits both sialyl Lewis x (Slex) expression on HT29 and ADP-induced activation of platelets through P-selectin- and GPIIb/IIIa-dependent mechanisms, confirmed by the corresponding antibody assay. CapNO inhibits platelet- or interleukin (IL)-1β-mediated adhesion between HT29 and endothelial cells, and micrometastatic formation in the lungs of immunocompetent syngeneic mouse models. CapNO have also shown the effects of vasodilation, anticoagulation, inhibition of matrix metalloproteinase-2 (MMP2) expression on cancer cells, and inhibition of cell adhesion molecules (CAMs) expression on vascular endothelium. Due to a series of the beneficial effects of CapNO, CTCs remain exposed to the hostile bloodstream environment and are vulnerable to death induced by shear stress and immune elimination. This new discovery provides a basis for CapNO used for cancer metastatic chemoprevention, and might suggest regulation of the CTCs bloodstream microenvironment as a new avenue for cancer metastatic prevention., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Aims: Maternal type 2 diabetes (T2D) can result in adverse pathological outcomes to both the mother and fetus. The present study aimed to investigate the pathological effects of maternal T2D on the gene expression patterns and functions of fetal human umbilical vein endothelial cells (HUVECs), a representative of fetal vascular cells., Methods: Cell proliferation, apoptosis, mitochondrial ROS production and cell cycle were measured using flowcytometry. Genome-wide expression was measured using Affymetrix microarray. Gene expression of CCND2, STAT1, ITGB8, ALDH2, and ADAMTS5 was measured using real-time PCR., Results: HUVECs derived from T2D mothers (T2D-HUVECs) showed elevated levels of mitochondrial superoxide anions, reduced cell proliferation, and increased apoptosis rates relative to HUVECs derived from healthy control mothers (C.HUVECs). In addition , T2D-HUVECs showed a decreased proportion of cells in G0/G1 and cell cycle arrest at the S phases relative to controls. Interestingly, microarray experiments revealed significant differences in genome-wide expression profiles between T2D-HUVECs and C.HUVECs. In particular, the analysis identified 90 upregulated genes and 42 downregulated genes. The upregulated genes CCND2, STAT1, ITGB8, ALDH2, and ADAMTS5 were validated as potential biomarkers for fetal endothelial dysfunction. Functional network analysis revealed that these genes are the important players that participate in the pathogenesis of endothelial dysfunction, which in turn influences the inflammatory response, cellular movement, and cardiovascular system development and function., Conclusion: Sustained alterations in the overall function of T2D-HUVEC and gene expression profiles provided insights into the role of maternal T2D on the pathophysiology of the fetal endothelial dysfunction.

Background/aims: Hydrazone and acylhydrazone derivatives, which are produced from aldehyde reacting with hydrazine or acylhydrazine, have been reported to exhibit antitumor activities. However, the angionenic effects of this kind of derivatives haven't been elucidated. Here, we synthesized 12 pyridoxal hydrazone and acylhydrazone compounds and investigated their antiangiogenic effects and the underlying mechanisms., Method: 3-(4,5-Dimethylthiazol-2-yl)-2, 5-dipheyltetrazolium bromide assay was used to screen the inhibitory effects of the synthesized compounds on endothelial cells (ECs) proliferation. The compound with best inhibitory effect was further evaluated with wound-healing assay and tube formation assay. Calcein-Am assay was carried out to determine the content of intracellular labile iron pool (LIP). Intracellular reduced glutathione (GSH) was determined by spectrophotometry. Flow cytometry was used to determine cell cycle and apoptosis., Results: Compound 10 (3-hydroxy-5-[hydroxymethyl]-2-methyl-pyridine-4-carbaldehyde-2-naphthalen-1-acetyl hydrazone) showed the best inhibitory effect on human umbilical vascular ECs proliferation, with IC50 value of 25.4 μmol/L. It not only inhibited wound-healing and tube formation of ECs, but also decreased the content of intracellular LIP and GSH. Furthermore, it arrested ECs cycle at S phase and induced cell apoptosis., Conclusions: Compound 10 exhibits antiangiogenic effects by reducing the content of intracellular LIP and GSH, and subsequently arresting cell cycle and inducing cell apoptosis., (© 2019 S. Karger AG, Basel.)

Objective: To investigate the effects of TNF-α-induced exosomes release on the biological behavior, metabolism, and bioenergetics of HUVECs., Methods: Exosomes were isolated from conditioned media of HUVECs by ultracentrifugation after treatment with or without TNF-α. HUVECs were treated with or without TNF-α, or different concentrations of exosomes isolated from conditioned media with or without TNF-α induction (T Exo and C Exo , respectively)., Results: The results showed that TNF-α significantly inhibited migration, tube formation, and increased apoptosis rate of HUVECs compared with controls. Furthermore, TNF-α-induced exosomes (T Exo ) rather than C Exo , indicated similar effects to inhibit migration, tube formation and promote endothelial apoptosis. Although TNF-α treatment did not show a statistical difference, T Exo significantly inhibited extracellular OCR compared with controls. T Exo could significantly inhibited intracellular OCR in a hypoxia condition. TNF-α significantly increased L-ECA compared with control cells, and T Exo showed similar stimulative effect on L-ECA., Conclusions: TNF-α-induced exosomes could significantly (a) change migration, tube formation, and apoptosis; (b) inhibit endothelial extracellular OCR and intracellular OCR (hypoxia); (c) increase glycolysis rate of the endothelial cells. These data provide new evidence for exploring endothelial behavior regulation using exosomes and their effects on endothelial metabolism and bioenergetics., (© 2018 John Wiley & Sons Ltd.)