Korean Circ J.  2009 Feb;39(2):57-65. 10.4070/kcj.2009.39.2.57.

Angiopoietin-1 Protects Endothelial Cells From Hypoxia-Induced Apoptosis via Inhibition of Phosphatase and Tensin Homologue Deleted From Chromosome Ten

• Lee SW
• Youn SW
• Kim TY
• Suh JW
• Koh GY
• Kwon YW
• Chae IH
• Park YB
• Kim HS

• KMID: 2297950
• DOI: http://doi.org/10.4070/kcj.2009.39.2.57

Abstract

BACKGROUND AND OBJECTIVES
Angiopoietin-1 (Ang1) is a regulator of blood vessel growth and maturation, and prevents radiation-induced or serum deprivation-induced apoptosis. Phosphatase and tensin homologue deleted from chromosome ten (PTEN), a well-known tumor suppressor, regulates cell cycle arrest and apoptosis. Hypoxia induces apoptosis by increasing the expression of PTEN. We hypothesized that Ang1 may regulate PTEN expression and, thus, reduce endothelial apoptosis under hypoxia in vitro and in vivo. Materials and
METHODS
In vitro, human umbilical vein endothelial cells (HUVECs) were treated with Ang1, and signaling pathways were investigated. In vivo, eight-week-old C57BL/6 mice were used for a hind limb ischemia model. Ang1 or normal saline was intramusculary injected. Blood flow was evaluated by a laser Doppler perfusion analyzer and tissue histology. RESULTS: The expression of PTEN was markedly upregulated in HUVECs after hypoxic stimulation, whereas Ang1 suppressed PTEN expression. Tie2-Fc, a soluble form of Tie2 (sTie2) that blocks Ang1, reversed the Ang1 effect on PTEN reduction under hypoxia. Ang1 inhibited the nuclear translocation of nuclear transcription factor-kB (NF-kB), a binding factor for the PTEN promoter and Foxo1. Hypoxia-induced p27 expression and apoptosis were also suppressed by Ang1. In the mouse hind limb ischemia model, we observed a high capillary density, numerous proliferating cells and diminished cell death in skeletal muscle tissue in the Ang1 injected group.
CONCLUSION
Ang1 enhanced endothelial cell survival by reducing apoptosis via PTEN down-regulation in HUVECs under hypoxia. Local injection of Ang1 significantly reduced apoptotic cells in vivo, and prevented limb loss for ischemic hind limb mice. Thus, Ang1 may be an effective therapeutic for protection from ischemic-endothelial cell injury.

Keyword

Angiopoietin-1; Hypoxia; Cell cycle; Ischemia

MeSH Terms

Angiopoietin-1
Animals
Anoxia
Apoptosis
Blood Vessels
Capillaries
Cell Cycle
Cell Cycle Checkpoints
Cell Death
Down-Regulation
Endothelial Cells
Extremities
Glycosaminoglycans
Human Umbilical Vein Endothelial Cells
Ischemia
Mice
Microfilament Proteins
Muscle, Skeletal
Perfusion
Angiopoietin-1
Glycosaminoglycans
Microfilament Proteins

Figure

• 

  Fig. 1 Ang1 reduced the expression of PTEN that was upregulated in HUVEC under hypoxic conditions. A: trypan blue exclusion assay showed that endothelial apoptosis increased in a time-dependent manner (*p<0.01). B: the expression of PTEN was upregulated under hypoxic conditions. Ang1 markedly reduced PTEN expression in HUVECs after 24 hours and 36 hours under hypoxia. C and D: RT-PCR and Western blot showed that Ang1 decreased hypoxia-induced PTEN expression. C: control, A: Ang1 (200 ng/mL), sTie2 (10 µg/mL). Ang1: angiopoietin-1, PTEN: phosphatase and tensin homologue deleted from chromosome ten, HUVECs: human umbilical vein en-dothelial cells, RT-PCR: reverse transcriptase-polymerase chain reaction.

• 

  Fig. 2 Ang1 inhibits p38MAPK and NF-kB activity in HUVECs under hypoxia. A: p38MAPK signaling pathways were tested by Western blot. p38 phosphorylation was strongly increased after 24 hours hypoxia, while Ang1 (200 ng/mL) clearly down regulated phospho-p38 (pp38). sTie2 (10 µg/mL) reversed Ang1 effect. Quantification of pp38 and IkB immunoblots from 2 independent experiments are shown at right. B: immunofluorescence staining for NF-kB in HUVECs after hypoxic stimulation for 24 hours. NF-kB stained red, the nucleus stained blue. C: control, A: Ang1 treatment (200 ng/mL), sTie2 (10 µg/mL). Ang1: angiopoietin-1, MAPK: mitogen activated protein kinase, NF-kB: nuclear factor-kappa B, HUVECs: human umbilical vein en-dothelial cells, IkB: inhibitory kappa B.

• 

  Fig. 3 Ang1 regulates forkhead transcription factor, Foxo1, but not Foxo3a. A: western blot using the nuclear fraction showed Foxo1 nuclear translocation. Ang1 (200 ng/mL) inhibited Foxo1 nuclear translocation after 24 hours hypoxia. There was no difference for Foxo3a translocation. B: western blot using whole cell lysates showed that the expression of Foxo1 was consistent. p-Foxo1 decreased under hypoxia, but increased with Ang1. C: control, A: Ang1 (200 ng/mL), sTie2 (10 µg/mL). Ang1: angiopoietin-1.

• 

  Fig. 4 Ang1 decreased the expression of p27 under hypoxia. A: the expression of p27 in HUVECs treated with 200 ng/mL Ang1 for 24 hours hypoxia was analyzed by RT-PCR. B: chromatin immunoprecipitation assay of p27 in specific genomic sequences from Ang1 treated HUVECs with anti-Foxo1 monoclonal antibody. Foxo1 binding activity in the p27 promoter region was strongly reduced by Ang1. C: control, A: Ang1 (200 ng/mL), sTie2 (10 µg/mL). Ang1: angiopoietin-1, HUVECs: human umbilical vein endothelial cells, RT-PCR: reverse transcriptase-polymerase chain reaction.

• 

  Fig. 5 Ang1 protected against hypoxia-induced HUVECs apoptosis. A: apoptotic fluorescence-activated cell sorting analysis measuring the hypo-diploid DNA contents, showing a significantly increased apoptotic fraction in the hypoxia group. However, Ang1 reduced endothelial apoptosis. B: trypan blue exclusion assay showed HUVECs viability at 24 hours and 48 hours after hypoxia with Ang1 treatment (*p<0.01). C: control, A: Ang1 (200 ng/mL), sTie2 (10 µg/mL). Ang1: angiopoietin-1, HUVECs: human umbilical vein endothelial cells, DNA: deoxyribonucleic acid.

• 

  Fig. 6 Ang1 showed greater efficacy to improve perfusion and reduce limb loss in mouse hind limb ischemia model. A: representative laser Doppler perfusion imaging (LDPI) analyses of hind limb blood perfusion for each group showing enhanced blood flow recovery in the Ang1 group. Local administration of 100 µg Ang1 by 3 injections into tight muscle (n=7) was evaluated by LDPI. B: representative figure of TUNEL staining at day 14. There were fewer TUNEL (+) cells (brown) in Ang1 injection group. C: quantitative analysis for apoptosis. TUNEL (+) cells were significantly decreased in Ang1 injection group (n=10; *p<0.05). D: immunofluorescence staining for PECAM-1 (green) in ischemic limb tissues at post-operative day 14. E: capillaries positive for PECAM-1 staining were significantly increased in Ang1 injection group (*p=0.016). F: immunofluorescene staining for BS-1 lectin and Ki67 in ischemic limb tissues at day 3, day 7 and day 14. There were more capillaries positive for BS-1 lectin (green) and Ki67 (red) in the Ang1 treatment group than in the saline group. G: Ki67/BS-1 lectin double positive cells were counted in 10 different microscopic fields. At least 3 different sections from each animal were used for counting (*p<0.05). Ang1: angiopoietin-1, TUNEL: terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling, PECAM: platelet endothelial cell adhesion molecule.

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