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• Correction

  Correction for: Sonic hedgehog signaling promotes angiogenesis of endothelial progenitor cells to improve pressure ulcers healing by PI3K/AKT/eNOS signaling

  Relevance score: 6.683496

  Jianhua Wang, Hongyan Zhan, Mingming Wang, Hua Song, Jianhua Sun, Gang Zhao

  Keywords: pressure ulcer, EPCs, SHH signaling, angiogenesis, PI3K/AKT/eNOS signaling

  Published in Aging on December 30, 2023

• Research Paper Volume 11, Issue 23 pp 11040-11053

  Sphingosine-1-phosphate promotes PDGF-dependent endothelial progenitor cell angiogenesis in human chondrosarcoma cells

  Relevance score: 8.055986

  Chao-Qun Wang, Chih-Yang Lin, Yuan-Li Huang, Shih-Wei Wang, Yan Wang, Bi-Fei Huang, Yu-Wei Lai, Shun-Long Weng, Yi-Chin Fong, Chih-Hsin Tang, Zhong Lv

  Keywords: S1P, PDGF-A, chondrosarcoma, angiogenesis, EPCs

  Published in Aging on December 6, 2019

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  The malignant bone tumors that are categorized as chondrosarcomas display a high potential for metastasis in late-stage disease. Higher-grade chondrosarcomas contain higher levels of expression of platelet-derived growth factor (PDGF) and its receptor. The phosphorylation of sphingosine by sphingosine kinase enzymes SphK1 and SphK2 generates sphingosine-1-phosphate (S1P), which inhibits human chondrosarcoma cell migration, while SphK1 overexpression suppresses lung metastasis of chondrosarcoma. We sought to determine whether S1P mediates levels of PDGF-A expression and angiogenesis in chondrosarcoma. Surprisingly, our investigations found that treatment of chondrosarcoma cells with S1P and transfecting them with SphK1 cDNA increased PDGF-A expression and induced angiogenesis of endothelial progenitor cells (EPCs). Ras, Raf, MEK, ERK and AP-1 inhibitors and their small interfering RNAs (siRNAs) inhibited S1P-induced PDGF-A expression and EPC angiogenesis. Our results indicate that S1P promotes the expression of PDGF-A in chondrosarcoma via the Ras/Raf/MEK/ERK/AP-1 signaling cascade and stimulates EPC angiogenesis.

  

  S1P increases PDGF-A expression and angiogenesis in human chondrosarcoma cells. (A, B) Chondrosarcoma cells were incubated with S1P (2.5–10 μM) for 24 h; PDGF-A expression was examined by qPCR and Western blot assays (n=4). (C, D) Chondrosarcoma cells were incubated with S1P for 24 h then stimulated with PDGF-A or IgG antibody (1 μg/ml) for 30 min. The conditioned medium (CM) was then collected and applied to endothelial progenitor cells (EPCs) (n=4). EPC migration and tube formation was measured. Results are expressed as the mean ± SEM. *p < 0.05 as compared with the control group; #p < 0.05 as compared with the S1P-treated group.

  
  
  

  

  Overexpression of SphK1 facilitates in PDGF-A expression and angiogenesis in human chondrosarcoma. (A, B) Chondrosarcoma cells were transfected with SphK1 cDNA; SphK1 and PDGF-A expression was examined by qPCR and Western blot assays (n=5). (C, D) The CM was applied to EPCs and analyses assessed migratory and tube formation activity (n=4). Results are expressed as the mean ± SEM. *p < 0.05 as compared with the vector group.

  
  
  

  

  The Ras and Raf pathways mediate S1P-promoted PDGF-A expression and angiogenesis. (A) Cells were pretreated for 30 min with manumycin A (10 μM) and GW5074 (10 μM), or transfected with Ras and Raf siRNAs then stimulated with S1P (10 μM). PDGF-A expression was examined by qPCR assays (n=5). (B, C) The CM was applied to EPCs and analyses assessed migratory and tube formation activity (n=4). (D) JJ012 cells were incubated with S1P; Ras and Raf activity was examined by Western blot assay (n=3). (E) JJ012 cells were pretreated with manumycin A for 30 min, then stimulated with S1P and Raf phosphorylation was examined (n=3). Results are expressed as the mean ± SEM. *p < 0.05 as compared with the control group; #p < 0.05 as compared with the S1P-treated group.

  
  
  

  

  The MEK and ERK pathways mediated S1P-promoted PDGF-A expression and angiogenesis. (A) Cells were pretreated for 30 min with PD98059 (10 μM) and U0126 (5 μM), or transfected with MEK and ERK siRNAs, then stimulated with S1P (10 μM). PDGF-A expression was examined by qPCR assays (n=5). (B, C) The CM was applied to EPCs and analyses assessed migratory and tube formation activity (n=4). (D) JJ012 cells were incubated with S1P; MEK and ERK phosphorylation was examined by Western blot assay (n=3). (E, F) JJ012 cells were pretreated with manumycin A, GW5074 and PD98059 for 30 min, then stimulated with S1P (10 μM). MEK and ERK phosphorylation was examined (n=3). Results are expressed as the mean ± SEM. *p < 0.05 as compared with the control group; #p < 0.05 as compared with the S1P-treated group.

  
  
  

  

  AP-1 is involved in S1P-facilitated PDGF-A expression and angiogenesis. (A) Cells were pretreated for 30 min with tanshinone IIA (3 μM), or transfected with c-Jun siRNA, then stimulated with S1P (10 μM). PDGF-A expression was examined by qPCR assays (n=5). (B, C) The CM was applied to EPCs and analyses assessed migratory and tube formation activity (n=4). (D) JJ012 cells were incubated with S1P (10 μM); c-Jun phosphorylation was examined by Western blot assay (n=3). (E) JJ012 cells were pretreated with manumycin A, GW5074, PD98059 and U0126 for 30 min, then stimulated with S1P (10 μM). The c-Jun phosphorylation was examined (n=3). (F, G) JJ012 cells were pretreated with Ras, Raf, MEK and ERK inhibitors or siRNAs, then stimulated with S1P (10 μM) and AP-1 Luciferase activity was examined (n=4). Results are expressed as the mean ± SEM. *p < 0.05 as compared with the control group; #p < 0.05 as compared with the S1P-treated group.

  
  
  

  

  Schematic diagram summarizes the mechanisms of S1P-promoted tumor angiogenesis in chondrosarcoma. S1P facilitates PDGF-A production via the Ras/Raf/MEK/ERK/AP-1 signaling pathway in human chondrosarcoma cells and subsequently induces EPC angiogenesis.

  
  
  

• Research Paper Volume 11, Issue 17 pp 6714-6733

  MeCP2 inhibits cell functionality through FoxO3a and autophagy in endothelial progenitor cells

  Relevance score: 7.878927

  Siyuan Zha, Zhen Li, Shuyan Chen, Fang Liu, Fei Wang

  Keywords: EPCs, autophagy, MeCP2, FoxO3a, epigenetics

  Published in Aging on September 2, 2019

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  Objectives: Autophagy is an evolutionarily conserved intracellular degradation mechanism in which cell constituents are phagocytosed to maintain cellular homeostasis. Forkhead box O 3a (FoxO3a) promotes autophagy to protect cells from environmental stress. Methylated CpG binding protein 2 (MeCP2) is a nuclear protein that binds DNA and represses transcription. However, the mechanism and interplay between FoxO3a and MeCP2 underlying endothelial progenitor cell (EPC) function are not fully understood.

  Results: In EPCs, MeCP2 overexpression attenuated autophagy and cell functionality, which were reversed by the autophagy activator rapamycin or co-transfection with FoxO3a. FoxO3a promoted cell function, which was reversed by the autophagy inhibitor chloroquine. Following MeCP2 overexpression, MeCP2 was found enriched on the FoxO3a promoter, resulting in promoter hypermethylation and enhanced H3K9 histone modification in nucleosomes of the FoxO3a promoter.

  Conclusions: MeCP2 attenuated cell functionality via DNA hypermethylation and histone modification of the FoxO3a promoter to inhibit FoxO3a transcription and autophagy.

  Materials and Methods: EPCs were isolated from human umbilical cord blood and treated with adenoviral vectors containing interference sequences. The effects and mechanism of MeCP2 and FoxO3a were analyzed by utilizing western blotting, cell counting kit-8, transwell plates, Matrigel, matrix adhesion, transmission electron microscopy, and chromatin immunoprecipitation.

  

  Identification of EPCs from human umbilical cord blood. (A) EPCs at passage 1 (P1) exhibited monolayer growth and cobblestone morphology after 2 weeks. (B) Cells were characterized by the immunofluorescence detection of CD31, CD34, VWF, VEGFR2, and CD133. (C) Flow cytometry revealed the positive expression rates of CD34, VEGFR2, and CD133. (D) Uptake of ac-LDL and binding of FITC-UEA-1 are characteristic functions of endothelial cells.

  
  
  

  

  Protein expression during replicative aging and the efficacy of adenoviral vectors containing interference sequences. (A) Senescence-associated beta-galactosidase (SA-β-gal) staining during P3, P12, and P21 confirmed the increasing rate of aging EPCs during replicative senescence. (B) Protein levels of MeCP2 and FoxO3a were detected by western blotting during replicative senescence. (C) Protein levels of MeCP2 and FoxO3a were detected by western blotting after transfection with Ad-sh-MeCP2 or Ad-sh-FoxO3a. (D) Protein levels of MeCP2 and FoxO3a were detected by western blotting after overexpression and silencing of MeCP2 or FoxO3a. (E) Efficacy of adenoviral vectors was confirmed under fluorescence microscopy. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control.

  
  
  

  

  FoxO3a promoted autophagy and cellular function. (A) LC3 II, Beclin1, p62, ATG5, and ATG7 protein levels were detected by western blotting after transfection with Ad-FoxO3a or Ad-sh-FoxO3a for 48 h. (B) Cell migration was evaluated by Transwell migration assays after transfection with Ad-FoxO3a or Ad-sh-FoxO3a for 48 h. (C) Cell adhesion ability was evaluated by matrix adhesion assays after transfection with Ad-FoxO3a or Ad-sh-FoxO3a for 48 h. (D) Angiogenic ability was evaluated by Matrigel assays after transfection with Ad-FoxO3a or Ad-sh-FoxO3a for 48 h. (E) Cell viability was evaluated with CCK-8 after transfection with Ad-FoxO3a or Ad-sh-FoxO3a for 48 h. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control.

  
  
  

  

  MeCP2 inhibited FoxO3a, autophagy, and cellular function. (A) Protein levels of FoxO3a, LC3 II, Beclin1, p62, ATG5, and ATG7 were detected by western blotting after transfection with Ad-MeCP2 or Ad-sh-MeCP2 for 48 h. (B) Cell migration was evaluated by Transwell migration assays after transfection with Ad-MeCP2 or Ad-sh-MeCP2 for 48 h. (C) Cell adhesion ability was evaluated by matrix adhesion assays after transfection with Ad-MeCP2 or Ad-sh-MeCP2 for 48 h. (D) Angiogenic ability was evaluated by Matrigel assays after transfection with Ad-MeCP2 or Ad-sh-MeCP2 for 48 h. (E) Cell viability was evaluated with CCK-8 after transfection with Ad-MeCP2 or Ad-sh-MeCP2 for 48 h. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control.

  
  
  

  

  FoxO3a promotes EPC functionality through autophagy. (A) LC3 II, Beclin1, p62, ATG5, and ATG7 protein levels were detected by western blotting after GFP, Ad-FoxO3a, Ad-FoxO3a + CQ, Ad-sh-FoxO3a, or Ad-sh-FoxO3a + Rapa. (B) Cell migration was evaluated by Transwell migration assays after treatment for 48 h. (C) Cell adhesion ability was evaluated by matrix adhesion assays after treatment for 48 h. (D) Angiogenic ability was evaluated by Matrigel assays after treatment for 48 h. (E) Cell viability was evaluated with CCK-8 after treatment for 48 h. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control. ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs. Ad-FoxO3a treatment. ^φP < 0.05, ^φφP < 0.01, and ^φφφP < 0.001 vs. Ad-sh-FoxO3a treatment.

  
  
  

  

  MeCP2 attenuates EPC functionality through autophagy. (A) FoxO3a, LC3 II, Beclin1, p62, ATG5, and ATG7 protein levels were detected by western blotting after GFP, Ad-MeCP2, Ad-MeCP2 + Rapa, Ad-sh-MeCP2, or Ad-sh-MeCP2 + CQ. (B) Cell migration was evaluated by Transwell migration assays after treatment for 48 h. (C) Cell adhesion ability was evaluated by matrix adhesion assays after treatment for 48 h. (D) Angiogenic ability was evaluated by Matrigel assays after treatment for 48 h. (E) Cell viability was evaluated with CCK-8 after treatment for 48 h. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control. ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs. Ad-MeCP2 treatment. ^φP < 0.05, ^φφP < 0.01, and ^φφφP < 0.001 vs. Ad-sh-MeCP2 treatment.

  
  
  

  

  MeCP2 inhibits autophagy and EPC function through FoxO3a. (A) Protein levels of MeCP2, FoxO3a, LC3 II, Beclin1, p62, ATG5, and ATG7 were detected by western blotting after transfection with Ad-MeCP2 or Ad-sh-MeCP2 or co-transfection with Ad-FoxO3a or Ad-sh-FoxO3a for 48 h. (B) EPC ultrastructure was imaged by TEM. The white arrow indicates autophagosomes. N, nucleus; M, mitochondria. (C) Cell migration was evaluated by Transwell migration assays after transfection or co-transfection for 48 h. (D) Cell adhesion ability was evaluated by matrix adhesion assay after transfection or co-transfection for 48 h. (E) Angiogenic ability was evaluated by Matrigel assays after transfection or co-transfection for 48 h. (F) Cell viability was evaluated with CCK-8 after transfection or co-transfection for 48 h. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control. ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 vs. Ad-MeCP2 treatment. ^φP < 0.05, ^φφP < 0.01, and ^φφφP < 0.001 vs. Ad-sh-MeCP2 treatment.

  
  
  

  

  MeCP2 inhibits FoxO3a transcription by promoting FoxO3a promoter methylation and enhancing H3K9 dimethylation. (A) Genome methylation was detected after MeCP2 overexpression. (B) DNA methylation was detected by BSP after MeCP2 overexpression. (C) ChIP of MeCP2 binding to the FoxO3a promoter after MeCP2 overexpression. (D) ChIP demonstrated H3K9me2 enrichment on the FoxO3a promoter after MeCP2 overexpression. (E) Proposed mechanism. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. control.

  
  
  

• Research Paper pp undefined-undefined

  Sonic hedgehog signaling promotes angiogenesis of endothelial progenitor cells to improve pressure ulcers healing by PI3K/AKT/eNOS signaling

  Relevance score: 6.1606913

  Jianhua Wang, Hongyan Zhan, Mingming Wang, Hua Song, Jianhua Sun, Gang Zhao

  Keywords: pressure ulcer, EPCs, SHH signaling, angiogenesis, PI3K/AKT/eNOS signaling

  Published in Aging on Invalid Date

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  Background: Pressure ulcer is a severe disease in the paralyzed and aging populations. Endothelial progenitor cells (EPCs) are able to regulate ulcer healing by modulating angiogenesis, but the molecular mechanism is still obscure. Sonic hedgehog (SHH) signaling contributes to angiogenesis in various diseases and has been identified to modulate EPCs function. Here, we aimed to explore the significance of SHH signaling in EPCs function during pressure ulcers.

  Methods: The EPCs were isolated and characterized by the expression of DiI-acLDL and bind fluorescein iso-thiocyanate UEA-1. Cell proliferation was detected by cell counting kit 8 (CCK-8). The DiI-acLDL and bind fluorescein iso-thiocyanate UEA-1 were analyzed by immunofluorescent analysis. The angiogenesis of EPCs was analyzed by tube formation assay. The pressure ulcers rat model was constructed, the wound injury was analyzed by H&E staining and angiogenesis was analyzed by the accumulation of CD31 based on immunofluorescent analysis.

  Results: The expression of patched-1 and Gli-1 were enhanced by SHH activator SAG but reduced by SHH inhibitor cyclopamine in the EPCsThe PI3K, Akt, eNOS expression and the Akt phosphorylation were induced by SAG, while the treatment of cyclopamine presented a reversed result. The proliferation and migration of EPCs were enhanced by SAG but repressed by cyclopamine or PI3K/AKT/eNOS signaling inhibitor Y294002, in which the co-treatment of Y294002 could reverse the effect of SAG.

  Conclusions: Thus, we found that SHH signaling activated angiogenesis properties of EPCs to improve pressure ulcers healing by PI3K/AKT/eNOS signaling. SHH signaling may serve as the potential targets for attenuating pressure ulcers.