Inhibition of oxygen induced retinal neovascularization by recombinant adeno-associated virus-polypyrimidine tract-binding protein-associated splicing factor intraocular injection in mice_Chinese Journal of Ocular Fundus Diseases

Authors：

TianFang ,  DongLijie , ZhouYu , 王飞 , 张晓敏 , 李筱荣

Tianjin Medical University Eye Hospital, Tianjin Medical University Eye Institute, The College of Optometry & Ophthalmology, Tianjin 300384, China;

Corresponding author：

DongLijie, Email: aitaomubang@126.com

Keywords：

Retinal neovascularization/prevention&control; Polypyrimidine tract-binding protein/drug effects; Animal experimentation

DOI：

10.3760/cma.j.issn.1005-1015.2014.05.019

Video：

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Objective To observe the inhibitory effect of intraocular injection of recombinant adeno-associated virus-polypyrimidine tract-binding protein-associated splicing factor (rAAV-PSF) in oxygen induced retinopathy mice model. Methods Eighteen C57BL/6J mice were divided into 3 groups randomly, including normal group, rAAV-PSF injection group, rAAV injection group. Western blot analysis was applied to detect the transfection expression level of PSF. The other 48 C57BL/6J mice were randomly divided into 4 groups: normal group, ischemia-induced retinopathy (OIR) group, rAAV-PSF treated OIR group, rAAV treated OIR group, 12 mice in each group. Placed all mice (excepted the mice in control group) in cages of (75±2)% oxygen concentration environment for 5 days then moved to a normal environment for 5 days to induced the OIR model. At the 12th day the OIR rAAV-PSF treated OIR group was intravitreal injected with 2 μl 5×10¹³ pfu/ml rAAV-PSF and the rAAV treated OIR group was treated with intravitreal injection of 2 μl 5×10¹³ pfu/ml rAAV. The mice in OIR group were left intact after moved out of oxygen cages. Five days after injection, the eyeballs were harvested and retinal sections were stained to count the nuclear of retinal endothelium cells. Western blot analysis was applied to detect the protein level of vascular endothelial growth factor (VEGF) in retina. Results There was a significant difference of the expression PSF between normal group and rAAV-PSF treated group (F=16.05,P=0.001). There was no significant difference of the PSF expression between normal group and rAAV treated group(F=16.05,P=0.890). There was a significant difference of the number of retinal endothelium cells nuclear between normal group and OIR group (F=101.00,P=0.007). There is a significant difference of the number of retinal endothelium cells nuclear between rAAV-PSF treated group and OIR group (F=101.00,P=0v002). There was no significant difference of the number of retinal endothelium cells nuclear between OIR rAAV treated group and OIR group (F=101.00,P=0.550). There was a significant difference of VEGF protein expression between normal group and OIR group (F=13.20,P=0.005), OIR group and rAAV treated OIR group (F=13.20,P=0.001). There was no difference of VEGF protein expression between OIR rAAV treated group and OIR group (F=13.20,P=0.071). Conclusion The rAAV-PSF vitreous injection can inhibit the expression of VEGF in OIR mice, hence to restrain the proliferation of neovascularization.

Citation： TianFang, DongLijie, ZhouYu. Inhibition of oxygen induced retinal neovascularization by recombinant adeno-associated virus-polypyrimidine tract-binding protein-associated splicing factor intraocular injection in mice. Chinese Journal of Ocular Fundus Diseases, 2014, 30(5): 504-508. doi: 10.3760/cma.j.issn.1005-1015.2014.05.019 Copy

1.	Wang XS,Ponnazhagan S,Srivastava A.Rescue and replication signals of the adeno-associated virus 2 genome[J].J Mol Biol,1995,250:573-580.
2.	Wang F,Rendahl KG,Manning WC,et al.AAV-mediated expression of vascular endothelial growth factor induces choroidal neovascularization in rat[J].Invest Ophthalmol Vis Sci,2003,44:781-790.
3.	Chen J, Michan S, Juan AM, et al. Neuronal sirtuin1 mediates retinal vascular regeneration in oxygen-induced ischemic retinopathy[J]. Angiogenesis, 2013, 16:985-992.
4.	Connor KM, Krah NM, Dennison RJ, et al.Quantification of oxygen-induced retinopathy in the mouse:a model of vessel loss, vessel regrowth and pathological angiogenesis[J]. Nat Prot, 2009, 4:1565-1573.
5.	Chen J, Joyal JS, Hatton CJ, et al. Propranolol inhibition of beta-adrenergic receptor does not suppress pathologic neovascularization in oxygen-induced retinopathy[J]. Invest Ophthalmol Vis Sci, 2012, 53:2968-2977.
6.	Penn JS, Madan A, Caldwell RB, et al. Vascular endothelial growth factor in eye disease[J]. Prog Retin Eye Res,2008,27:331-371.
7.	Xia XB, Xiong SQ, Song WT, et al. Inhibition of retinal neovascularization by siRNA targeting VEGF(165)[J]. Mol Vis,2008,14:1965-1973.
8.	Dye BT, Patton JG. An RNA recognition motif (RRM) is required for the localization of PTB-associated splicing factor (PSF) to subnuclear speckles[J]. Exp Cell Res, 2001, 263:131-144.
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12.	Urban RJ, Bodenburg YH, Wood TG. NH2 terminus of PTB-associated splicing factor binds to the porcine P450scc IGF-I response element[J]. Am J Physiol Endocrinol Metab, 2002, 283:423-427.
13.	Urban RJ, Bodenburg Y, Kurosky A, et al. Polypyrimidine tract-binding protein-associated splicing factor is a negative regulator of transcriptional activity of the porcine p450scc insulin-like growth factor response element[J]. Mol Endocrinol, 2000, 14:774-782.
14.	Bhisitkul RB, Robinson GS, Moulton RS, et al. An antisense oligodeoxynucleotide against vascular endothelial growth factor in a nonhuman primate model of iris neovascularization[J]. Arch Ophthalmol, 2005, 123:214-219.
15.	Luthra S NR, Marques LE, Chwa M, et al. Evaluation of in Vitro Effects of Bevacizumab (Avastin) on Retinal Pigment Epithelial, Neurosensory Retinal, and Microvascular Endothelial Cells[J]. Retina, 2006, 26:512-518.
16.	Saishin Y, Saishin Y, Takahashi K, et al. VEGF-TRAP(R1R2) suppresses choroidal neovascularization and VEGF-induced breakdown of the blood-retinal barrier[J]. J Cell Physiol, 2003, 195:241-248.
17.	Nguyen QD, Shah SM, Hafiz G, et al. A phase I trial of an IV-administered vascular endothelial growth factor trap for treatment in patients with choroidal neovascularization due to age-related macular degeneration[J]. Ophthalmology, 2006,113:1521-1522.
18.	Auricchio A, Behling KC, Maguire AM, et al. Inhibition of retinal neovascularization by intraocular viral-mediated delivery of anti-angiogenic agents[J]. Mol Ther,2002,6:490-494.
19.	Bainbridge JW, Mistry A, De Alwis M, et al. Inhibition of retinal neovascularisation by gene transfer of soluble VEGF receptor sFlt-1[J]. Gene Ther, 2002, 9:320-326.
20.	Deng WT, Yan Z, Dinculescu A, et al. Adeno-associated virus-mediated expression of vascular endothelial growth factor peptides inhibits retinal neovascularization in a mouse model of oxygen-induced retinopathy[J]. Hum Gene Ther, 2005,16:1247-1254.
21.	Pan H, Nguyen NQ, Yoshida H, et al. Molecular targeting of antiangiogenic factor 16K hPRL inhibits oxygen-induced retinopathy in mice[J]. Invest Ophthalmol Vis Sci,2004,45:2413-2419.
22.	Li S,Li T,Luo Y,et al.Retro-orbital injection of FITC-dextran is an effective and economical method for observing mouse retinal vessels[J].Mol Vis,2011,17:3566-3573.

1. Wang XS,Ponnazhagan S,Srivastava A.Rescue and replication signals of the adeno-associated virus 2 genome[J].J Mol Biol,1995,250:573-580.
2. Wang F,Rendahl KG,Manning WC,et al.AAV-mediated expression of vascular endothelial growth factor induces choroidal neovascularization in rat[J].Invest Ophthalmol Vis Sci,2003,44:781-790.
3. Chen J, Michan S, Juan AM, et al. Neuronal sirtuin1 mediates retinal vascular regeneration in oxygen-induced ischemic retinopathy[J]. Angiogenesis, 2013, 16:985-992.
4. Connor KM, Krah NM, Dennison RJ, et al.Quantification of oxygen-induced retinopathy in the mouse:a model of vessel loss, vessel regrowth and pathological angiogenesis[J]. Nat Prot, 2009, 4:1565-1573.
5. Chen J, Joyal JS, Hatton CJ, et al. Propranolol inhibition of beta-adrenergic receptor does not suppress pathologic neovascularization in oxygen-induced retinopathy[J]. Invest Ophthalmol Vis Sci, 2012, 53:2968-2977.
6. Penn JS, Madan A, Caldwell RB, et al. Vascular endothelial growth factor in eye disease[J]. Prog Retin Eye Res,2008,27:331-371.
7. Xia XB, Xiong SQ, Song WT, et al. Inhibition of retinal neovascularization by siRNA targeting VEGF(165)[J]. Mol Vis,2008,14:1965-1973.
8. Dye BT, Patton JG. An RNA recognition motif (RRM) is required for the localization of PTB-associated splicing factor (PSF) to subnuclear speckles[J]. Exp Cell Res, 2001, 263:131-144.
9. Dong L, Zhang X, Fu X, et al. PTB-associated splicing factor (PSF) functions as a repressor of STAT6-mediated Ig epsilon gene transcription by recruitment of HDAC1[J]. J Biol Chem, 2011, 286:3451-3459.
10. Lukong KE, Huot ME, Richard S. BRK phosphorylates PSF promoting its cytoplasmic localization and cell cycle arrest[J]. Cell Signal, 2009, 21:1415-1422.
11. Urban RJ, Bodenburg Y. PTB-associated splicing factor regulates growth factor-stimulated gene expression in mammalian cells[J]. Am J Physiol Endocrinol Metab, 2002, 283:794-798.
12. Urban RJ, Bodenburg YH, Wood TG. NH2 terminus of PTB-associated splicing factor binds to the porcine P450scc IGF-I response element[J]. Am J Physiol Endocrinol Metab, 2002, 283:423-427.
13. Urban RJ, Bodenburg Y, Kurosky A, et al. Polypyrimidine tract-binding protein-associated splicing factor is a negative regulator of transcriptional activity of the porcine p450scc insulin-like growth factor response element[J]. Mol Endocrinol, 2000, 14:774-782.
14. Bhisitkul RB, Robinson GS, Moulton RS, et al. An antisense oligodeoxynucleotide against vascular endothelial growth factor in a nonhuman primate model of iris neovascularization[J]. Arch Ophthalmol, 2005, 123:214-219.
15. Luthra S NR, Marques LE, Chwa M, et al. Evaluation of in Vitro Effects of Bevacizumab (Avastin) on Retinal Pigment Epithelial, Neurosensory Retinal, and Microvascular Endothelial Cells[J]. Retina, 2006, 26:512-518.
16. Saishin Y, Saishin Y, Takahashi K, et al. VEGF-TRAP(R1R2) suppresses choroidal neovascularization and VEGF-induced breakdown of the blood-retinal barrier[J]. J Cell Physiol, 2003, 195:241-248.
17. Nguyen QD, Shah SM, Hafiz G, et al. A phase I trial of an IV-administered vascular endothelial growth factor trap for treatment in patients with choroidal neovascularization due to age-related macular degeneration[J]. Ophthalmology, 2006,113:1521-1522.
18. Auricchio A, Behling KC, Maguire AM, et al. Inhibition of retinal neovascularization by intraocular viral-mediated delivery of anti-angiogenic agents[J]. Mol Ther,2002,6:490-494.
19. Bainbridge JW, Mistry A, De Alwis M, et al. Inhibition of retinal neovascularisation by gene transfer of soluble VEGF receptor sFlt-1[J]. Gene Ther, 2002, 9:320-326.
20. Deng WT, Yan Z, Dinculescu A, et al. Adeno-associated virus-mediated expression of vascular endothelial growth factor peptides inhibits retinal neovascularization in a mouse model of oxygen-induced retinopathy[J]. Hum Gene Ther, 2005,16:1247-1254.
21. Pan H, Nguyen NQ, Yoshida H, et al. Molecular targeting of antiangiogenic factor 16K hPRL inhibits oxygen-induced retinopathy in mice[J]. Invest Ophthalmol Vis Sci,2004,45:2413-2419.
22. Li S,Li T,Luo Y,et al.Retro-orbital injection of FITC-dextran is an effective and economical method for observing mouse retinal vessels[J].Mol Vis,2011,17:3566-3573.

Chinese Journal of Ocular Fundus Diseases

Inhibition of oxygen induced retinal neovascularization by recombinant adeno-associated virus-polypyrimidine tract-binding protein-associated splicing factor intraocular injection in mice

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