scholarly journals P53/DRAM/ autophagy - a new target for improving the therapeutic effect of anti-VEGF on intraocular neovascularization

2020 ◽  
Author(s):  
Yi Wang ◽  
Yao Yang ◽  
Rong Li ◽  
Binghui Wu ◽  
Huiqin Lu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Migration ◽  
Therapeutic Effect ◽  
Vascular Endothelial Cells ◽  
Tube Formation ◽  
Control Group ◽  
Vascular Endothelial ◽  
Autophagy Inhibitor ◽  
Anti Vegf ◽  
Autophagy Inhibition

Abstract Backgroud Recurrence of intraocular neovascularization is a major clinical problem. Anti-VEGF drugs are the main treatment for intraocular neovascularization currently. However, anti-VEGF drugs can activate endothelial autophagy and weaken the therapeutic effect. This study aims to elucidate the effect and mechanism of anti-VEGF drugs on autophagy of vascular endothelial cells. Methods RF/6A cells were randomly divided into five groups: The control group, hypoxia group (1% O2、5% CO2、94% N2), anti-VEGF group(group1:Ranibizumab 100ug/ml; group2: Aflibercept, 400ug/ml; group3: Conbercept, 100ug/ml) and autophagy inhibition group(3-MA or CQ) which was corresponding to anti-VEGF group. Autophagy-related proteins were examined by Western blot. RFP-GFP-LC3 was used to detect autophagy and autophagic flow. CCK-8 assay was used to detect cell proliferation. Flow cytometry and Tunel was used to detect cell apoptosis. Cell migration and tube formation were assessed by wound assay and matrix method, respectively. Results Ranibizumab and Conbercept can triger autophagy in hypoxia condition in RF/6A cells, while Aflibercept can inhibit autophagy. Conbercept combined with autophagy inhibitor (3-MA or CQ) could inhibit cell migration and tube formation of RF/6A cells more effectively in hypoxia condition. For mechanism, p53 and DRAM proteins paly an important role in Conbercept induced autophagy. Inhibition of P53 can suppressed the autophagy induced by Conbercept. Conclusion Ranibizumab and Conbercept can triger the autophagy of vascular endothelial cells while Aflibercept can inhibit it. The combination of ranibizumab/ Concept and autophagy inhibitor can significantly inhibit the formation of angiogenesis in vitro. The mechanism of autophagy activation is related to the activation of p53 / DRAM pathway.

CYLD regulates angiogenesis by mediating vascular endothelial cell migration

Blood ◽  
2010 ◽  
Vol 115 (20) ◽  
pp. 4130-4137 ◽  
Author(s):  
Jinmin Gao ◽  
Lei Sun ◽  
Lihong Huo ◽  
Min Liu ◽  
Dengwen Li ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Vascular Endothelial Cells ◽  
Vascular Endothelial Cell ◽  
Tube Formation ◽  
Endothelial Cell Migration ◽  
Vascular Endothelial

Cylindromatosis (CYLD) is a deubiquitinase that was initially identified as a tumor suppressor and has recently been implicated in diverse normal physiologic processes. In this study, we have investigated the involvement of CYLD in angiogenesis, the formation of new blood vessels from preexisting ones. We find that knockdown of CYLD expression significantly impairs angiogenesis in vitro in both matrigel-based tube formation assay and collagen-based 3-dimensional capillary sprouting assay. Disruption of CYLD also remarkably inhibits angiogenic response in vivo, as evidenced by diminished blood vessel growth into the angioreactors implanted in mice. Mechanistic studies show that CYLD regulates angiogenesis by mediating the spreading and migration of vascular endothelial cells. Silencing of CYLD dramatically decreases microtubule dynamics in endothelial cells and inhibits endothelial cell migration by blocking the polarization process. Furthermore, we identify Rac1 activation as an important factor contributing to the action of CYLD in regulating endothelial cell migration and angiogenesis. Our findings thus uncover a previously unrecognized role for CYLD in the angiogenic process and provide a novel mechanism for Rac1 activation during endothelial cell migration and angiogenesis.

scholarly journals Effects of IL-1β and TNF-α on the Expression of P311 in Vascular Endothelial Cells and Wound Healing in Mice

2020 ◽  
Vol 11 ◽  
Author(s):  
Daijun Zhou ◽  
Tengfei Liu ◽  
Song Wang ◽  
Weifeng He ◽  
Wei Qian ◽  
...  
Keyword(s):  
Wound Healing ◽  
Endothelial Cells ◽  
Cell Migration ◽  
Protein Expression ◽  
Vascular Endothelial Cells ◽  
Control Group ◽  
Vascular Endothelial ◽  
Expression Levels ◽  
Relative Expression ◽  
Tnf Α

ObjectiveThis study aimed to define the role of interleukine-1β (IL-1β) and tumor necrosis factor-α (TNF-α) in the expression of P311 in vascular endothelial cells (VECs) and in wound healing.MethodsDAPI staining, a CCK-8 assay, cell migration assay, and an angiogenesis assay were used to assess the effects exerted by TNF-α and IL-1β at various concentrations on morphology, proliferation, migration, and angiogenesis of VECs. Western blot (WB) and reverse transcription-polymerase chain reaction (RT-PCR) models were employed to observe the effects exerted by proteins related to the nuclear factor-kappa B (NF-κB) signaling pathway and P311 mRNA expression. Bioinformatics analysis was performed on the binding sites of P311 and NF-κB. Finally, to investigate the effects of IL-1β and TNF-α on wound healing and the length of new epithelium in mice, we established a full-thickness wound defect model in mice. Immunohistochemistry was used to measure changes in P311, proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF), CD31 (platelet endothelial cell adhesion molecule-1, PECAM-1/CD31), as well as other related proteins.ResultsWhen levels of TNF-α and IL-1β were both 20 ng/ml, their effects on cell proliferation, cytoskeleton protein expression, cell migration, and angiogenesis were the greatest (P < 0.05). IL-1β and TNF-α at moderate concentrations effectively promoted P311 mRNA and p-NF-κB protein expression (P < 0.05), while p-NF-K b protein expression was decreased (P < 0.05). Luciferase assays showed that P311 expression was also relatively greater when stimulated at moderate concentrations (P < 0.05), while relative expression was significantly lower when the p-NF-K b inhibitor CAPE was added (P < 0.05). On 7-day wound healing rate comparison, the control, IL-1β, IL-1βab, TNF-α, and TNF-αab groups were 18, 37, 35, 39, and 36%, respectively, while control group + P311 siRNA was 31% (P < 0.05). New epithelial length, granulation tissue thickness, and number of blood vessels trends were also the same. In the control group, P311 showed lower relative expression levels than the others (P < 0.05). P311 relative expression levels trended as follows: control group > IL-1βab > IL-1β > TNF-αab > TNF-α (P < 0.05).ConclusionWhen IL-1β and TNF-α concentrations are moderate, they effectively promote the proliferation, expression, migration, and angiogenesis of VECs, possibly by promoting the expression of the NF-K b pathway and thereby promoting the expression of P311. In vitro experiments on mice suggest that P311 effectively promotes wound healing, and its mechanism may be closely related to PCNA, CD31, and VEGF.

scholarly journals The Use of Fiber-Reinforced Scaffolds Cocultured with Schwann Cells and Vascular Endothelial Cells to Repair Rabbit Sciatic Nerve Defect with Vascularization

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Hongyang Gao ◽  
Yang You ◽  
Guoping Zhang ◽  
Feng Zhao ◽  
Ziyi Sha ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Sciatic Nerve ◽  
Nerve Fiber ◽  
Schwann Cells ◽  
Vascular Endothelial Cells ◽  
Neurological Function ◽  
Control Group ◽  
Vascular Endothelial ◽  
Fiber Reinforced ◽  
3D Scaffolds

To explore the feasibility of biodegradable fiber-reinforced 3D scaffolds with satisfactory mechanical properties for the repair of long-distance sciatic nerve defect in rabbits and effects of vascularized graft in early stage on the recovery of neurological function, Schwann cells and vascular endothelial cells were cocultured in the fiber-reinforced 3D scaffolds. Experiment group which used prevascularized nerve complex for the repair of sciatic nerve defect and control group which only cultured with Schwann cells were set. The animals in both groups underwent electromyography to show the status of the neurological function recovery at 4, 8, and 16 weeks after the surgery. Sciatic nerve regeneration and myelination were observed under the light microscope and electron microscope. Myelin sheath thickness, axonal diameter, and number of myelinated nerve fiber were quantitatively analyzed using image analysis system. The recovery of foot ulcer, the velocity of nerve conduction, the number of regenerating nerve fiber, and the recovery of ultrastructure were increased in the experimental group than those in the control group. Prevascularized tissue engineered fiber-reinforced 3D scaffolds for the repair of sciatic nerve defects in rabbits can effectively promote the recovery of neurological function.

scholarly journals Tricyclic antidepressant amitriptyline inhibits autophagic flux and prevents tube formation in vascular endothelial cells

2018 ◽  
Vol 124 (4) ◽  
pp. 370-384 ◽  
Author(s):  
Yinglu Guan ◽  
Xiang Li ◽  
Michihisa Umetani ◽  
Krishna M. Boini ◽  
Pin‐Lan Li ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Tube Formation ◽  
Tricyclic Antidepressant ◽  
Autophagic Flux ◽  
Vascular Endothelial

scholarly journals Protective Effects of Oxymatrine on Vascular Endothelial Cells from High-Glucose-Induced Cytotoxicity by Inhibiting the Expression of A2B Receptor

2018 ◽  
Vol 45 (2) ◽  
pp. 558-571 ◽  
Author(s):  
Yun Yi ◽  
Yulin Shen ◽  
Qin Wu ◽  
Jingan Rao ◽  
Shu Guan ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Viability ◽  
Western Blotting ◽  
High Glucose ◽  
Vascular Endothelial Cells ◽  
Control Group ◽  
Vascular Endothelial ◽  
A2b Receptor ◽  
High Glucose Group ◽  
Glucose Group

Background/Aims: Diabetes mellitus (DM) has become an increasingly epidemic metabolic disease. Vascular endothelial cells play a key role in developing the cardiovascular complications of DM. The A2B receptor is expressed in vascular endothelial cells, and may help regulate the function of endothelial cells. The aim of this study was to investigate the protective effects of oxymatrine (OMT) on human umbilical vein endothelial cells (HUVECs) from high glucose-induced cytotoxicity. Methods: Homology modeling and molecular docking analysis were used to detect the binding sites between the adenosine A2B receptor and OMT. HUVECs were cultured with control (5.5 mM) or elevated glucose (22.2 mM) in the presence or absence of 3 µM OMT or A2B siRNA for 3 days. The MTS cell viability assay was used to measure the toxicity of high glucose on HUVECs and the protective effect of OMT or A2B siRNA. The expression of the adenosine A2B receptor and CCL5 in HUVECs was detected with real-time quantitative PCR (qPCR) and Western blotting methods in each group. Levels of IL-1β and TNF-α were measured using an enzyme-linked immunosorbent assay (ELISA) kit, and the concentration of NO was detected with the nitrate reductase method. Monocyte chemotactic activity in each group was detected using Transwell chambers. Furthermore, the phosphorylation of p38 and ERK1/2 in each group was observed through the Western blotting method. Results: Homology modeling and molecular docking analysis showed that OMT contains well-fitted binding sites to the A2B receptor. After chronic culture at high glucose, the rate of cell viability was significantly lower than that of the control group. After co-treatment with OMT or A2B siRNA, cell viability was significantly increased compared with the high-glucose group. The results from real-time quantitative RT-PCR (qRT-PCR) and Western blotting indicated that high glucose could increase the expression of A2B receptors in HUVECs, an effect that was inhibited by OMT. In addition, the results revealed that the expression of CCL5, IL-1β and TNF-α was increased in the high-glucose group, and that the NO produced by HUVECs decreased due to hyperglycemia; however, co-culture with OMT or A2B siRNA abolished these effects. Meanwhile, the chemotaxis activity of monocytes to HUVECs cultured in high-glucose medium was enhanced 2.59-fold compared to the control cells. However, the inflammatory reactions in HUVECs were completely relieved by co-treatment with OMT or A2B siRNA. Moreover, the phosphorylation of p38 and ERK1/2 in HUVECs in the high-glucose group was significantly higher than that of the control group; these effects were reversed after co-treatment with OMT or A2B siRNA. Conclusion: OMT may protect the HUVECs from high glucose-induced cytotoxicity through inhibitting the expression of A2B receptor and inflammatory factors as well as decreasing the phosphorylation of p38 and ERK1/2.

scholarly journals Involvement of Acid-Sensing Ion Channel 1a Contribute to The Effect of Extracellular Acidosis on Vascular Endothelialcell Damage of Henoch-Schönlein Purpura Patients

2020 ◽  
Author(s):  
Jin-Kun Wang ◽  
Yan Bo ◽  
Qi-lian Zhou ◽  
Li-ping Yuan
Keyword(s):  
Endothelial Cells ◽  
Ion Channel ◽  
Vascular Endothelial Cells ◽  
Binding Activity ◽  
Control Group ◽  
Vascular Endothelial ◽  
Henoch Schonlein Purpura ◽  
Protein Expressions ◽  
Cytoskeleton Protein ◽  
Schonlein Purpura

Abstract Background: Henoch-Schönlein purpura (HSP) is a common kind of systematic vasculitis in children characterized by rash, joint pain,abdominal symptoms and renal disease,the detail pathogenesis of HSP has not been elucidated.Acid-sensing ion channels (ASICs) is a proton-gated sodium selective channel that belongs to Degenerin /epithelial Sodium Channel(DEG/ENaC) superfamily,previous research found the expressions of ASIC1a and ASIC3 in the vascular endothelial cells of HSPThis study aims to investigate the molecular mechanisms of silencing of acid-sensing ion channel 1a(ASIC1a) protects the vascular endothelial cells from Henoch-Schonlein purpura(HSP) patients.Findings:Human dermal microvascular endothelial cells ( HDMVEC) were cultured in vitro, siRNA sequences were designed for the coding region of human ASIC1a gene and HDMVEC cells were transfected with recombinant lentivirus( LV) -sh-ASIC1a. The control group ( NC group) without virus transfection and LV-sh-ASIC1a transfection group ( si-ASIC1a group) were set up. The expression of transfixed ASIC1a gene was detected by RT-PCR. After virus transfection 72 h, serum IgA1from HSP patients and serum IgA1 of normal children were added into HDMVEC cells. ASIC1a and cytoskeleton protein ( sm-α f-actin, MLCK) mRNA and protein expressions were detected by real-time PCR and Western blotting Methods.The binding activity of NF- κB with DNA in HDMVEC was determined by electrophoretic mobility shift assay (EMSA).The whole-cell patch-clamp technique was used to record the current changes and electrophysiological characteristics of ASICs in HDMVEC.Cytoskeleton protein ( sm-α f-actin, MLCK) mRNA and protein expressions in the group of si-ASIC1a group were significantly increased compared with the NC control group( P＜0.01). The HSP serum and silencing of ASIC1a had no significant effect on the binding activity of NF-κB with DNA in HDMVEC. Compared with the NC control group, the ASICS current and calcium overload of the si-ASIC1A group were reduced( P＜0.01) ．Conclusions: Silencing ASIC1a can protect HSP vascular endothelial cell injury by inhibiting ASIC1-related calcium influx and reducing the overload of calcium ,not the NF- κB signaling pathway.

scholarly journals Rat epidermal stem cells promote the angiogenesis of full-thickness wound

2020 ◽  
Author(s):  
Shaobin Huang ◽  
Zhicheng Hu ◽  
Peng Wang ◽  
Yi zhang ◽  
Xiaoling Cao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Wound Healing ◽  
Endothelial Cells ◽  
Cell Suspension ◽  
Vascular Endothelial Cells ◽  
Control Group ◽  
Vascular Endothelial ◽  
Epidermal Stem Cells ◽  
Full Thickness ◽  
Full Thickness Wound

Abstract Background: Full-thickness wounds are a serious problem which badly affects patients’ life quality and also become the difficult problem for clinicians. Stem cells have great prospects in the treatment of wounds. Our previous experiments proved that autologous basal cell suspension can promote wound healing, and there are epidermal stem cells (ESCs) in basal cell suspension. We then conducted experiments to explore the effect of ESCs on full-thickness wound. Methods: In our study, the rat ESCs were isolated and expanded, and transfected with lentivirus to stably express EGFP. Experimental rats were randomly divided into 2 groups, in the ESCs group, the rat ESCs were sprayed on the Full-thickness wounds of the rats, while in control group, sprayed the PBS on the wound. Wound healing and neovascularization were then evaluated. Colonization, division and differentiation of ESCs on the wound were discovered by immunofluorescence.Results: The result suggested that rat ESCs can colonize, divide and proliferate in the wound. What’s more, the rat ESCs around blood vessels can differentiate into vascular endothelial cells and form a lumen-like structure. Compared with the control group, spraying the rat ESCs on the wound bed can promote angiogenesis and accelerate wound healing. Conclusions: Our study proved that rat ESCs were safe and effective for treating full-thickness wounds, and under certain conditions, ESCs can differentiate into vascular endothelial cells to promote angiogenesis and wound healing.

scholarly journals Tim-3 promotes tube formation and decreases tight junction formation in vascular endothelial cells

2020 ◽  
Vol 40 (10) ◽  
Author(s):  
Yizi Cong ◽  
Xingmiao Wang ◽  
Suxia Wang ◽  
Guangdong Qiao ◽  
Yalun Li ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tight Junction ◽  
Immune Escape ◽  
Vascular Endothelial Cells ◽  
Tumour Progression ◽  
Umbilical Vein ◽  
Tube Formation ◽  
In Vitro Assays ◽  
Vegf Receptor ◽  
Vascular Endothelial

Abstract As a negative immune checkpoint molecule, T-cell immunoglobulin domain and mucin domain containing molecule-3 (Tim-3) has been found to serve a crucial role in immune escape and tumour progression. Previous studies have reported that Tim-3 is important to endothelial cells and it has also been demonstrated to be involved in numerous types of human diseases, including melanoma, lymphoma, rickettsial infection and atherosclerosis; however, its exact mechanism of action remains largely unknown. In the present study, Tim-3 was overexpressed in vascular endothelial human lung microvascular endothelial cells (HMVECs) and human umbilical vein endothelial cells (HUVECs), and in vitro assays were used to determine that Tim-3 promoted cell proliferation, migration, invasion and tube formation through activating cyclin D1 (CCND1), Ras homolog gene family member A and vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2). Additionally, Tim-3 decreased tight junction (TJ) formation and the transepithelial resistance (TER) of endothelial cells by decreasing the expression levels of TJ protein 2, Occludin and claudin 1 (CLND1). In conclusion, these findings suggested that Tim-3 may exert a positive role in angiogenesis and a negative role in TJ formation in vascular endothelial cells, which may provide novel strategies for the treatment of Tim-3-associated diseases.

Sign in / Sign up

Export Citation Format

Share Document