Adenoviral 15-lipoxygenase-1 gene transfer inhibits hypoxia-induced proliferation of retinal microvascular endothelial cells in vitro

Ying Yan, Tao He, Ying Shen, Xiao Chen, Bo Diao, Zhi Li, Qing Liu, Yi Qiao Xing

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

AIM: To investigate whether 15-Lipoxygenase-1 (15-LOX-1) plays an important role in the regulation of angiogenesis, inhibiting hypoxia-induced proliferation of retinal microvascular endothelial cells (RMVECs) and the underlying mechanism. METHODS: Primary RMVECs were isolated from the retinas of C57/BL6J mice and identified by an evaluation for FITC-marked CD31. The hypoxia models were established with the Bio-bag and evaluated with a blood-gas analyzer. Experiments were performed using RMVECs treated with and without transfer Ad-15-LOX-1 or Ad-vector both under hypoxia and normoxia condition at 12, 24, 48, 72 hours. The efficacy of the gene transfer was assessed by immunofluorescence staining. Cells proliferation was evaluated by the CCK-8 method. RNA and protein expressions of 15-LOX-1, VEGF-A, VEGFR-2, eNOs and PPAR-r were analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot. RESULTS: Routine evaluation for FITC-marked CD31 showed that cells were pure. The results of blood-gas analysis showed that when the cultures were exposed to hypoxia for more than 2 hours, the Po2 was 4.5 to 5.4 Kpa. We verified RMVECs could be infected with Ad-15-LOX-1 Ad-vector or via Fluorescence microscopy. CCK-8 analysis revealed that the proliferative capacities of RMVECs in hypoxic group were significantly higher at each time point than they were in normoxic group (P <0.05). In a hypoxic condition, the proliferative capacities of RMVECs in 15-LOX-1 group were significantly inhibited (P <0.05). Real-time RT-PCR analysis revealed that the expressions of VEGF-A, VEGF-R2 and eNOs mRNA increased in hypoxia group compared with normoxia group (P <0.01). However, the expressions of 15-LOX-1, PPAR-r mRNA decreased in hypoxia group compared with normoxia group (P <0.01). It also showed that in a hypoxic condition, the expressions of VEGF-A, VEGF-R2 and eNOs mRNA decreased significantly in 15-LOX-1 group compared with hypoxia group (P <0.01). However, 15-LOX-1 and PPAR-r mRNA increased significantly in 15-LOX-1 group compared with hypoxia group (P <0.01). There was no significant difference of the mRNA expressions between vector group and hypoxia group (P >0.05). Western blot analysis revealed that the expressions of relative proteins were also ranked in that order. CONCLUSION: Our results suggested that 15-LOX-1 and PPAR-r might act as a negative regulator of retinal angiogenesis. And the effect of 15-LOX-1 overexpression is an anti-angiogenic factor in hypoxia-induced retinal neovascularization (RNV). Overexpression 15-LOX-1 on RMVECs of hypoxia-induced RNV blocked signaling cascades by inhibiting hypoxia-induced increases in VEGF family. PPAR-r effect on VEGFR2 could be an additional mechanism whereby 15-LOX-1 inhibited the hypoxia-induced RNV. Copyright International Journal of Ophthalmology Press.

Original languageEnglish (US)
Pages (from-to)562-569
Number of pages8
JournalInternational Journal of Ophthalmology
Volume5
Issue number5
DOIs
StatePublished - 2012

Fingerprint

Arachidonate 15-Lipoxygenase
Lipoxygenase
Endothelial Cells
Retinal Neovascularization
Peroxisome Proliferator-Activated Receptors
Genes
Sincalide
Fluorescein-5-isothiocyanate
Vascular Endothelial Growth Factor A
Western Blotting
Cell Hypoxia
Vascular Endothelial Growth Factor Receptor-2
Blood Gas Analysis
Hypoxia
In Vitro Techniques
Angiogenesis Inducing Agents
Fluorescence Microscopy
Reverse Transcription
Fluorescent Antibody Technique
Retina

Keywords

  • 15-Lipoxygenase-1
  • Hypoxia
  • Retinal neovascularization
  • Retinalmicrovascular endothelial cells

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Adenoviral 15-lipoxygenase-1 gene transfer inhibits hypoxia-induced proliferation of retinal microvascular endothelial cells in vitro. / Yan, Ying; He, Tao; Shen, Ying; Chen, Xiao; Diao, Bo; Li, Zhi; Liu, Qing; Xing, Yi Qiao.

In: International Journal of Ophthalmology, Vol. 5, No. 5, 2012, p. 562-569.

Research output: Contribution to journalArticle

Yan, Ying ; He, Tao ; Shen, Ying ; Chen, Xiao ; Diao, Bo ; Li, Zhi ; Liu, Qing ; Xing, Yi Qiao. / Adenoviral 15-lipoxygenase-1 gene transfer inhibits hypoxia-induced proliferation of retinal microvascular endothelial cells in vitro. In: International Journal of Ophthalmology. 2012 ; Vol. 5, No. 5. pp. 562-569.
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abstract = "AIM: To investigate whether 15-Lipoxygenase-1 (15-LOX-1) plays an important role in the regulation of angiogenesis, inhibiting hypoxia-induced proliferation of retinal microvascular endothelial cells (RMVECs) and the underlying mechanism. METHODS: Primary RMVECs were isolated from the retinas of C57/BL6J mice and identified by an evaluation for FITC-marked CD31. The hypoxia models were established with the Bio-bag and evaluated with a blood-gas analyzer. Experiments were performed using RMVECs treated with and without transfer Ad-15-LOX-1 or Ad-vector both under hypoxia and normoxia condition at 12, 24, 48, 72 hours. The efficacy of the gene transfer was assessed by immunofluorescence staining. Cells proliferation was evaluated by the CCK-8 method. RNA and protein expressions of 15-LOX-1, VEGF-A, VEGFR-2, eNOs and PPAR-r were analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot. RESULTS: Routine evaluation for FITC-marked CD31 showed that cells were pure. The results of blood-gas analysis showed that when the cultures were exposed to hypoxia for more than 2 hours, the Po2 was 4.5 to 5.4 Kpa. We verified RMVECs could be infected with Ad-15-LOX-1 Ad-vector or via Fluorescence microscopy. CCK-8 analysis revealed that the proliferative capacities of RMVECs in hypoxic group were significantly higher at each time point than they were in normoxic group (P <0.05). In a hypoxic condition, the proliferative capacities of RMVECs in 15-LOX-1 group were significantly inhibited (P <0.05). Real-time RT-PCR analysis revealed that the expressions of VEGF-A, VEGF-R2 and eNOs mRNA increased in hypoxia group compared with normoxia group (P <0.01). However, the expressions of 15-LOX-1, PPAR-r mRNA decreased in hypoxia group compared with normoxia group (P <0.01). It also showed that in a hypoxic condition, the expressions of VEGF-A, VEGF-R2 and eNOs mRNA decreased significantly in 15-LOX-1 group compared with hypoxia group (P <0.01). However, 15-LOX-1 and PPAR-r mRNA increased significantly in 15-LOX-1 group compared with hypoxia group (P <0.01). There was no significant difference of the mRNA expressions between vector group and hypoxia group (P >0.05). Western blot analysis revealed that the expressions of relative proteins were also ranked in that order. CONCLUSION: Our results suggested that 15-LOX-1 and PPAR-r might act as a negative regulator of retinal angiogenesis. And the effect of 15-LOX-1 overexpression is an anti-angiogenic factor in hypoxia-induced retinal neovascularization (RNV). Overexpression 15-LOX-1 on RMVECs of hypoxia-induced RNV blocked signaling cascades by inhibiting hypoxia-induced increases in VEGF family. PPAR-r effect on VEGFR2 could be an additional mechanism whereby 15-LOX-1 inhibited the hypoxia-induced RNV. Copyright International Journal of Ophthalmology Press.",
keywords = "15-Lipoxygenase-1, Hypoxia, Retinal neovascularization, Retinalmicrovascular endothelial cells",
author = "Ying Yan and Tao He and Ying Shen and Xiao Chen and Bo Diao and Zhi Li and Qing Liu and Xing, {Yi Qiao}",
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TY - JOUR

T1 - Adenoviral 15-lipoxygenase-1 gene transfer inhibits hypoxia-induced proliferation of retinal microvascular endothelial cells in vitro

AU - Yan, Ying

AU - He, Tao

AU - Shen, Ying

AU - Chen, Xiao

AU - Diao, Bo

AU - Li, Zhi

AU - Liu, Qing

AU - Xing, Yi Qiao

PY - 2012

Y1 - 2012

N2 - AIM: To investigate whether 15-Lipoxygenase-1 (15-LOX-1) plays an important role in the regulation of angiogenesis, inhibiting hypoxia-induced proliferation of retinal microvascular endothelial cells (RMVECs) and the underlying mechanism. METHODS: Primary RMVECs were isolated from the retinas of C57/BL6J mice and identified by an evaluation for FITC-marked CD31. The hypoxia models were established with the Bio-bag and evaluated with a blood-gas analyzer. Experiments were performed using RMVECs treated with and without transfer Ad-15-LOX-1 or Ad-vector both under hypoxia and normoxia condition at 12, 24, 48, 72 hours. The efficacy of the gene transfer was assessed by immunofluorescence staining. Cells proliferation was evaluated by the CCK-8 method. RNA and protein expressions of 15-LOX-1, VEGF-A, VEGFR-2, eNOs and PPAR-r were analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot. RESULTS: Routine evaluation for FITC-marked CD31 showed that cells were pure. The results of blood-gas analysis showed that when the cultures were exposed to hypoxia for more than 2 hours, the Po2 was 4.5 to 5.4 Kpa. We verified RMVECs could be infected with Ad-15-LOX-1 Ad-vector or via Fluorescence microscopy. CCK-8 analysis revealed that the proliferative capacities of RMVECs in hypoxic group were significantly higher at each time point than they were in normoxic group (P <0.05). In a hypoxic condition, the proliferative capacities of RMVECs in 15-LOX-1 group were significantly inhibited (P <0.05). Real-time RT-PCR analysis revealed that the expressions of VEGF-A, VEGF-R2 and eNOs mRNA increased in hypoxia group compared with normoxia group (P <0.01). However, the expressions of 15-LOX-1, PPAR-r mRNA decreased in hypoxia group compared with normoxia group (P <0.01). It also showed that in a hypoxic condition, the expressions of VEGF-A, VEGF-R2 and eNOs mRNA decreased significantly in 15-LOX-1 group compared with hypoxia group (P <0.01). However, 15-LOX-1 and PPAR-r mRNA increased significantly in 15-LOX-1 group compared with hypoxia group (P <0.01). There was no significant difference of the mRNA expressions between vector group and hypoxia group (P >0.05). Western blot analysis revealed that the expressions of relative proteins were also ranked in that order. CONCLUSION: Our results suggested that 15-LOX-1 and PPAR-r might act as a negative regulator of retinal angiogenesis. And the effect of 15-LOX-1 overexpression is an anti-angiogenic factor in hypoxia-induced retinal neovascularization (RNV). Overexpression 15-LOX-1 on RMVECs of hypoxia-induced RNV blocked signaling cascades by inhibiting hypoxia-induced increases in VEGF family. PPAR-r effect on VEGFR2 could be an additional mechanism whereby 15-LOX-1 inhibited the hypoxia-induced RNV. Copyright International Journal of Ophthalmology Press.

AB - AIM: To investigate whether 15-Lipoxygenase-1 (15-LOX-1) plays an important role in the regulation of angiogenesis, inhibiting hypoxia-induced proliferation of retinal microvascular endothelial cells (RMVECs) and the underlying mechanism. METHODS: Primary RMVECs were isolated from the retinas of C57/BL6J mice and identified by an evaluation for FITC-marked CD31. The hypoxia models were established with the Bio-bag and evaluated with a blood-gas analyzer. Experiments were performed using RMVECs treated with and without transfer Ad-15-LOX-1 or Ad-vector both under hypoxia and normoxia condition at 12, 24, 48, 72 hours. The efficacy of the gene transfer was assessed by immunofluorescence staining. Cells proliferation was evaluated by the CCK-8 method. RNA and protein expressions of 15-LOX-1, VEGF-A, VEGFR-2, eNOs and PPAR-r were analyzed by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot. RESULTS: Routine evaluation for FITC-marked CD31 showed that cells were pure. The results of blood-gas analysis showed that when the cultures were exposed to hypoxia for more than 2 hours, the Po2 was 4.5 to 5.4 Kpa. We verified RMVECs could be infected with Ad-15-LOX-1 Ad-vector or via Fluorescence microscopy. CCK-8 analysis revealed that the proliferative capacities of RMVECs in hypoxic group were significantly higher at each time point than they were in normoxic group (P <0.05). In a hypoxic condition, the proliferative capacities of RMVECs in 15-LOX-1 group were significantly inhibited (P <0.05). Real-time RT-PCR analysis revealed that the expressions of VEGF-A, VEGF-R2 and eNOs mRNA increased in hypoxia group compared with normoxia group (P <0.01). However, the expressions of 15-LOX-1, PPAR-r mRNA decreased in hypoxia group compared with normoxia group (P <0.01). It also showed that in a hypoxic condition, the expressions of VEGF-A, VEGF-R2 and eNOs mRNA decreased significantly in 15-LOX-1 group compared with hypoxia group (P <0.01). However, 15-LOX-1 and PPAR-r mRNA increased significantly in 15-LOX-1 group compared with hypoxia group (P <0.01). There was no significant difference of the mRNA expressions between vector group and hypoxia group (P >0.05). Western blot analysis revealed that the expressions of relative proteins were also ranked in that order. CONCLUSION: Our results suggested that 15-LOX-1 and PPAR-r might act as a negative regulator of retinal angiogenesis. And the effect of 15-LOX-1 overexpression is an anti-angiogenic factor in hypoxia-induced retinal neovascularization (RNV). Overexpression 15-LOX-1 on RMVECs of hypoxia-induced RNV blocked signaling cascades by inhibiting hypoxia-induced increases in VEGF family. PPAR-r effect on VEGFR2 could be an additional mechanism whereby 15-LOX-1 inhibited the hypoxia-induced RNV. Copyright International Journal of Ophthalmology Press.

KW - 15-Lipoxygenase-1

KW - Hypoxia

KW - Retinal neovascularization

KW - Retinalmicrovascular endothelial cells

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