Autophagy In Angiogenesis Research Articles

Treponema pallidum membrane protein Tp47 promotes angiogenesis through ROS-induced autophagy.

Pathological angiogenesis is an important manifestation of syphilis, but the underlying mechanism of Treponema pallidum subspecies pallidum (T.pallidum)-induced angiogenesis is poorly understood. The objective of this study is to investigate the role and related mechanism of the T.pallidum membrane protein Tp47 in angiogenesis. The proangiogenic activity of recombinant T.pallidum membrane protein Tp47 in human umbilical vein endothelial cells (HUVECs) was assessed by tube formation assay, three-dimensional angiogenesis analysis and experiments with a zebrafish embryo model. The effects of mitochondrial ROS and NADPH oxidase on intracellular ROS induced by Tp47 were further investigated. Furthermore, the levels of autophagy-related proteins and autophagic flux were measured. Finally, the role of ROS-induced autophagy in angiogenesis was studied. Tp47 promoted tubule formation and the formation of angiogenic sprouts in vitro. In addition, a significant increase in the number of subintestinal vessel branch points in zebrafish injected with Tp47 was observed using a zebrafish embryo model. Tp47 also significantly increased intracellular ROS levels in a dose-dependent manner. Tp47-induced tube formation and angiogenic sprout formation were effectively prevented by the ROS inhibitor NAC. In addition, Tp47 enhanced the production of mitochondrial ROS and expression of the NADPH oxidase-related proteins Nox2 and Nox4. The production of mitochondrial ROS and intracellular ROS was reduced by the NADPH oxidase inhibitors DPI and apocynin. Furthermore, Tp47 significantly increased expression of the autophagy-related proteins P62 and Beclin 1 and the LC3-II/LC3-I ratio and promoted an increase in autophagic flux, which could be effectively rescued by coincubation with the ROS inhibitor NAC. Further intervention with the autophagy inhibitor BafA1 significantly inhibited tube formation and angiogenic sprout formation. Tp47-induced NADPH oxidase enhanced intracellular ROS production via mitochondrial ROS and promoted angiogenesis through autophagy mediated by ROS. These findings may contribute to our understanding of pathological angiogenesis in syphilis.

Role of autophagy induced by arecoline in angiogenesis of oral submucous fibrosis

ObjectivesTo detect the expression of protein light chain 3 (LC3) and p62-SQSTM1 (p62) in the lamina propria of oral submucous fibrosis (OSF) and to determine the association of autophagy with OSF. To investigate the role of autophagy in angiogenesis of human umbilical vein endothelial cells (HUVECs) and to assess whether this effect was induced by arecoline. MethodsLC3 and p62 expression was detected in OSF tissue through immunohistochemistry (IHC). Transmission electron microscopy (TEM) and Western blot were used to investigate the expression of autophagy in HUVECs. The role of autophagy in angiogenesis in HUVECs was investigated using the Matrigel assay. Results1: LC3 expression was upregulated in OSF samples. In contrast, p62 was downregulated in early and intermediate stages but upregulated in advanced stages of OSF. 2: HUVECs treated with arecoline exhibited increased autophagosomes, LC3 expression and reduced p62 expression, when co-treated with chloroquine (CQ), which is a specific autophagy inhibitor, revealed the opposite trend. 3: Autophagy inhibited angiogenesis in HUVECs. ConclusionsOur findings suggest that arecoline induces autophagy in HUVECs. The high level of autophagy could reduce cell viability and inhibit angiogenesis in HUVECs, potentially promoting the development of OSF.

Role of autophagy in angiogenesis of retinal vascular endothelial cells under hypoxia condition

Objective To study the role of autophagy in proliferation, migration and tube formation of retinal vascular endothelial cells (RVECs) under the condition of hypoxia in vitro. Methods Mouse RVECs were isolated from 50 C57BL/6J mice primarily cultured using explant culture method, and the cells were identified by immunofluorescence of CD34.Well cultured RVECs were divided into normal control group, hypoxia control group and hypoxia+ 3-methyladenine (3-MA) group.The cells in the normal control group were cultured in the normal environment for 24 hours, and the cells in the hypoxia control group were cultured 1% O2 environment for 24 hours, and the cells in the hypoxia+ 3-MA group were pretreated with 5 mmol/L 3-MA for 4 hours and then exposed to 1% O2 environment for 24 hours.Microtubule-related protein 1 light chain 3 (LC3-Ⅱ/LC3-Ⅰ) and Beclin-1 protein contents in the cells were detected by Western blot analysis; the ultrastructure of autophagosome was examined under the transmission electron microscope.The proliferation, migration and tube formation of the cells were detected by Click-iTEdU kit, scratch assay and matrigel, respectively. Results Primarily culture cells grew well with the cobblestone-like appearance 5-7 days after culture and showed positive response for CD34.The autophagosome number was increased in the hypoxia control group compared with the hypoxia+ 3-MA group and normal control group.The LC3-Ⅱ/LC3-Ⅰ ratio was 0.243±0.030, 0.658±0.032 and 0.405±0.095; the relative expression of Beclin-1 protein in the cells was 0.260±0.040, 0.650±0.071 and 0.461±0.089; the proliferation rate of the cells was (45.93±6.39)%, (22.74±2.35)% and (24.12±3.59)%; the scratch healing rate of the cells was (36.02±5.84)%, (57.26±11.98)% and (18.16±9.73)%; the number of tube formation was 29.20±6.10, 41.40±4.04 and 22.00±2.92 in the normal control group, hypoxia control group and hypoxia+ 3-MA group, respectively, with significant differences among the groups (F=35.86, 23.53, 34.28, 21.12, 23.27; all at P<0.01). Compared with the normal control group, the ratio of LC3-Ⅱ/LC3-Ⅰ and the expression of Beclin-1 protein, scratch healing rate and the number of tube formation were significantly increased, and the proliferation rate was significantly reduced in the hypoxia control group (all at P<0.05). Compared with the hypoxia control group, the ratio of LC3-Ⅱ/LC3-Ⅰ and the expression of Beclin-1 protein, scratch healing rate and the number of tube formation were significantly decreased in the hypoxia+ 3-MA group. Conclusions Hypoxia environment activates autophagy of mouse RVECs, which enhances the migration and tube formation abilities of the cells.3-MA inhibits the angiogenesis abilities of mouse RVECs. Key words: Autophagy; Angiogenesis; Hypoxia; Retinal neovascularization; Ischemic retinopathy; Cells, cultured; Mice, inbred C57BL

Role of Autophagy in Angiogenesis Induced by a High-Glucose Condition in RF/6A Cells

Purpose: To study the effect of autophagy on vitality, migration, and tube formation of RF/6A cells under the condition of <smlcap>D</smlcap>-glucose. Methods: Cultured RF/6A cells were randomly divided into 4 groups (control, low glucose, high glucose, and high glucose with 3-methyladenine [3-MA]). Autophagy-related proteins (Atg7, p62, and LC3) were monitored. Cell vitality, cell migration, tube formation, reactive oxygen species (ROS) production, and apoptosis were assessed. Results: Cell vitality significantly decreased and cell migration and tube formation significantly increased in the high-glucose group (p < 0.05). Pretreatment with 3-MA significantly increased cell viability and inhibited cell migration and tube formation (p < 0.05). ROS production increased in the high-glucose group and decreased in the high-glucose with N-acetylcysteine (NAC) group (p < 0.05). The level of apoptosis increased in the high-glucose group, while it was reduced in the high-glucose with 3-MA group. Conclusion: Autophagy maybe participates in the formation of retinal neovascularization induced by high glucose.

T7 peptide inhibits angiogenesis via downregulation of angiopoietin-2 and autophagy.

Angiogenesis is required for the invasion, metastasis and chemoresistance of tumor cells. In addition to vascular endothelial cell growth factor (VEGF), angiopoietin-2 (Ang2) is considered to be a promising target for anti-angiogenic therapy. The T7 peptide, an active fragment of full-length tumstatin [the noncollagenous 1 domain of the type IV collagen α3 chain, α3(IV)NC1], has equivalent anti-angiogenic activity to that of full-length tumstatin. This study aimed to explore the mechanisms of the T7 peptide in the regulation of Ang2 expression in endothelial cells (ECs) as well as inhibition of angiogenesis and invasion of hepatocellular carcinoma cells. We also examined the role of autophagy in angiogenesis. Human umbilical vein endothelial cells (HUVECs) were incubated with endothelial cell medium (ECM)-2 in a hypoxia chamber to mimic hypoxic conditions. The recombinant T7 peptide inhibited the cell viability, tube formation and induced apoptosis of the HUVECs. The T7 peptide downregulated the protein expression of Ang2 by inhibiting phosphorylation of AKT under hypoxic conditions. The migration of HUVECs and invasion of HepG2 cells were inhibited by the T7 peptide via inhibition of Ang2 expression. EC autophagy was induced by the T7 peptide. Inhibition of autophagy enhanced the anti‑angiogenic activity of the T7 peptide by increasing EC apoptosis. In vivo, immunohistochemistry of VE-cadherin and CD31 showed that angiogenesis was decreased significantly by the T7 peptide in a nude mouse xenogeneic tumor model. In conclusion, the T7 peptide inhibited angiogenesis and exerted its antitumor effects by inhibition of Ang2, phosphorylation of AKT and matrix metalloproteinase-2 (MMP-2) regulated by Ang2. Furthermore, inhibition of autophagy may significantly enhance the anti-angiogenic activity of the T7 peptide.

Role of autophagy in angiogenesis in aortic endothelial cells

Angiogenesis plays critical roles in the recovery phase of ischemic heart disease and peripheral vascular disease. An increase in autophagy is protective under hypoxic and chronic ischemic conditions. In the present study we determined the role of autophagy in angiogenesis. 3-Methyladenine (3-MA) and small interfering RNA (siRNA) against ATG5 were used to inhibit autophagy induced by nutrient deprivation of cultured bovine aortic endothelial cells (BAECs). Assays of BAECs tube formation and cell migration revealed that inhibition of autophagy by 3-MA or siRNA against ATG5 reduced angiogenesis. In contrast, induction of autophagy by overexpression of ATG5 increased BAECs tube formation and migration. Additionally, inhibiting autophagy impaired vascular endothelial growth factor (VEGF)-induced angiogenesis. However, inhibition of autophagy did not alter the expression of pro-angiogenesis factors such as VEGF, platelet-derived growth factor, or integrin αV. Furthermore, autophagy increased reactive oxygen species (ROS) formation and activated AKT phosphorylation. Inhibition of autophagy significantly decreased the production of ROS and activation of AKT but not of extracellular regulated kinase, whereas overexpression of ATG5 increased cellular ROS production and AKT activation in BAECs. Inhibition of AKT activation or ROS production significantly decreased the tube formation induced by ATG5 overexpression. Here we report a novel observation that autophagy plays an important role in angiogenesis in BAECs. Induction of autophagy promotes angiogenesis while inhibition of autophagy suppresses angiogenesis, including VEGF-induced angiogenesis. ROS production and AKT activation might be important mechanisms for mediating angiogenesis induced by autophagy. Our findings indicate that targeting autophagy may provide an important new tool for treating cardiovascular disease.

The Role Of Autophagy In Angiogenesis In Aortic Endothelial Cells

Angiogenesis plays critical roles in the pathogenesis of ischemic heart diseases and peripheral vascular diseases. The objective of this study is to examine the role of autophagy in angiogenesis. Using cultured bovine aortic endothelial cells (BAEC), autophagy was induced either by nutrient‐deprivation or by overexpression of ATG5. Both treatments significantly increased autophagosome formation and increased expression of light chain3 II (LC3II). Interestingly, both nutrient‐deprivation and ATG5 overexpression increased EC tube formation, migration as well as wound‐healing process. To determine whether autphagy is required process for angiogenesis, EC were treated with a specific auotphagy inhibitor 3‐methyladenine (3‐MA) or Beclin‐1 siRNA to suppress autophagy activation. Interestingly, 3‐MA or Beclin‐1 siRNA greatly decreased EC tube formation, migration and wound‐healing under nutrient‐deprived conditions, and blocked VEGF‐induced tube formation and migration. Autophagy inhibition did not affect the expression of VEGF, PDGF and integrin αV. Furthermore, autophagy inhibition decreased AKT activation and ROS production, two important contributors to angiogenesis. In conclusion, we report for the first time that autophagy appears to be essential for angiogenesis and that targeting autophagy provides an important new route in treating cardiovascular diseases.