Reduction of experimental diabetic vascular leakage by delivery of angiostatin with a recombinant adeno-associated virus vector.
OBJECTIVE
To evaluate the efficacy of recombinant adeno-associated virus (rAAV) vector expressing mouse angiostatin (Kringle domains 1 to 4) in reducing retinal vascular leakage in an experimental diabetic rat model.


METHODS
rAAV-angiostatin was delivered by intravitreal injection to the right eyes of Sprague-Dawley rats. As a control, the contralateral eye received an intravitreal injection of rAAV-lacZ. Gene delivery was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). Diabetes was induced by intravenous injection of streptozotocin (STZ). Vascular permeability changes were evaluated by extravascular albumin accumulation and leakage of intravenous-injected fluorescein isothiocynate-bovine serum albumin (FITC-BSA). Effects of rAAV-angiostatin on expression of vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), occludin, and phospho-p42/p44 MAP kinase in retina tissue were analyzed by western blotting.


RESULTS
The rAAV-angiostatin injections led to sustained angiostatin gene expression in retina as confirmed by RT-PCR, and reduced extravascular albumin accumulation in STZ-induced diabetic retina. Further, rAAV-angiostatin significantly decreased intravascularly injected FITC-BSA leakage at 5 days (p=0.001), 10 days (p<0.001), and 15 days (p=0.001) after STZ-induced diabetes, as compared to the control eyes receiving rAAV-lacZ. Expression of VEGF and phosphorylation of p42/p44 MAP kinase in retina was reduced by rAAV-angiostatin at day 1 (p=0.043 for both VEGF and phospho-p42/p44 MAP kinase) after STZ-induced diabetes compared with rAAV-lacZ eyes. rAAV-angiostatin reduced retinal occludin loss at 10 days after STZ-induced diabetes (n=5, p=0.041). There was no significant difference in retinal PEDF expression between eyes injected with rAAV-angiostatin and rAAV-lacZ.


CONCLUSIONS
Intravitreal delivery of rAAV-angiostatin reduces vascular leakage in an STZ-induced diabetic model. This effect is associated with a reduction in the retinal occludin loss induced by diabetes and downregulation of retinal VEGF and phosphor-p42/p44 MAP kinase expression. This gene transfer approach may reduce diabetic macular edema, providing protection in diabetic patients at risk for macular edema.
Shyong
Mong-Ping
MP
Institute of Clinical Medicine, National Yang-Ming University, Taipei and Su-Ao Veterans Hospital, I-Lan, Taiwan. t1201001@ms2.kntech.com.tw <t1201001@ms2.kntech.com.tw>
Lee
Fenq-Lih
FL
Kuo
Ping-Chang
PC
Wu
Ai-Ching
AC
Cheng
Huey-Chung
HC
Chen
Show-Li
SL
Tung
Tao-Hsin
TH
Tsao
Yeou-Ping
YP
D043168:Angiostatins / Q000235:genetics*; D000818:Animals; D001813:Blood-Retinal Barrier; D002199:Capillary Permeability; D000229:Dependovirus / Q000235:genetics*; D003921:Diabetes Mellitus, Experimental; D003930:Diabetic Retinopathy / Q000378:metabolism / Q000503:physiopathology*; D015536:Down-Regulation; D005136:Eye Proteins / Q000378:metabolism; D018014:Gene Transfer Techniques; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009414:Nerve Growth Factors / Q000378:metabolism; D062793:Occludin; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D011995:Recombination, Genetic; D012160:Retina / Q000378:metabolism; D012171:Retinal Vessels / Q000503:physiopathology*; D015843:Serpins / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D014822:Vitreous Body; D000099174:Pigment Epithelium-Derived Factor
D043168:Angiostatins; D005136:Eye Proteins; D008565:Membrane Proteins; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D009414:Nerve Growth Factors; D062793:Occludin; D015843:Serpins; D042461:Vascular Endothelial Growth Factor A; D000099174:Pigment Epithelium-Derived Factor; C568986:Ocln protein, mouse; C568987:Ocln protein, rat