Anti-angiogenic effects of a mutant endostatin: a new prospect for treating retinal and choroidal neovascularization.
OBJECTIVE
Pathological fundus angiogenesis is a major cause of vision loss in retina diseases. Endostatin, a C-terminal fragment of collagen XVIII, is an endogenous anti-angiogenic protein. The present study aimed to investigate the in vitro and in vivo anti-angiogenic properties of two proteins: an N-terminal H1D/H3D mutant endostatin (M-ES) and a polyethylene glycol propionaldehyde (PEG) covalent M-ES (PEG-M-ES).


METHODS
M-ES and PEG-M-ES properties were characterized in vitro using a zinc ion binding assay and a stability test. Activity assays, including migration, proliferation, and tube formation assays, were performed with human retinal microvascular endothelial cells (HRMECs) and human umbilical vein endothelial cells (HUVECs). Mouse oxygen-induced retinopathy (OIR) and choroidal neovascularization (CNV) models were used to evaluate in vivo anti-angiogenic effects. In addition, a rabbit model was used to study the retinal pharmacokinetic profile following an intravitreal injection.


RESULTS
The results indicated that the H1D/H3D mutations of endostatin reduced the zinc binding capacity of M-ES and facilitated PEG covalent binding. PEG-M-ES was more stable and persisted longer in the retina compared with M-ES. The in vitro studies demonstrated that M-ES and PEG-M-ES inhibited HRMEC and HUVEC proliferation, migration, and tube formation more efficiently than ES. In vivo, a single intravitreal injection of M-ES and PEG-M-ES significantly decreased neovascularization in both the OIR and CNV animal models.


CONCLUSIONS
The present study demonstrated for the first time that PEG-M-ES exhibits a long-term inhibitory effect on neovascularization in vitro and in vivo. These data suggest that PEG-M-ES may represent an innovative therapeutic strategy to prevent fundus neovascularization.
Key Laboratory of Vision Loss and Restoration, Ministry of Education, Beijing Key Laboratory for the Diagnosis and Treatment of Retinal and Choroid Diseases, Department of Ophthalmology, Peking University People's Hospital, Beijing, People's Republic of China.
Yujing
Y
Bai
Key Laboratory of Vision Loss and Restoration, Ministry of Education, Beijing Key Laboratory for the Diagnosis and Treatment of Retinal and Choroid Diseases, Department of Ophthalmology, Peking University People's Hospital, Beijing, People's Republic of China.
Min
M
Zhao
Clinical Epidemiology & Biostatistics, Peking University People's Hospital, Beijing, People's Republic of China.
Chunfang
C
Zhang
Key Laboratory of Vision Loss and Restoration, Ministry of Education, Beijing Key Laboratory for the Diagnosis and Treatment of Retinal and Choroid Diseases, Department of Ophthalmology, Peking University People's Hospital, Beijing, People's Republic of China.
Shanshan
S
Li
Key Laboratory of Vision Loss and Restoration, Ministry of Education, Beijing Key Laboratory for the Diagnosis and Treatment of Retinal and Choroid Diseases, Department of Ophthalmology, Peking University People's Hospital, Beijing, People's Republic of China.
Yun
Y
Qi
Key Laboratory of Vision Loss and Restoration, Ministry of Education, Beijing Key Laboratory for the Diagnosis and Treatment of Retinal and Choroid Diseases, Department of Ophthalmology, Peking University People's Hospital, Beijing, People's Republic of China.
Bin
B
Wang
Key Laboratory of Vision Loss and Restoration, Ministry of Education, Beijing Key Laboratory for the Diagnosis and Treatment of Retinal and Choroid Diseases, Department of Ophthalmology, Peking University People's Hospital, Beijing, People's Republic of China.
Lvzhen
L
Huang
Key Laboratory of Vision Loss and Restoration, Ministry of Education, Beijing Key Laboratory for the Diagnosis and Treatment of Retinal and Choroid Diseases, Department of Ophthalmology, Peking University People's Hospital, Beijing, People's Republic of China.
Xiaoxin
X
Li
D020533:Angiogenesis Inhibitors / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology*; D000818:Animals; D001483:Base Sequence; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D020256:Choroidal Neovascularization / Q000517:prevention & control*; D043169:Endostatins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D042783:Endothelial Cells / Q000187:drug effects / Q000502:physiology; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000502:physiology; D006801:Humans; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009154:Mutation; D009389:Neovascularization, Pathologic / Q000517:prevention & control; D018919:Neovascularization, Physiologic / Q000187:drug effects; D011092:Polyethylene Glycols / Q000737:chemistry; D011817:Rabbits; D012160:Retina / Q000378:metabolism / Q000473:pathology; D012164:Retinal Diseases / Q000473:pathology / Q000503:physiopathology; D015861:Retinal Neovascularization / Q000517:prevention & control*; D012171:Retinal Vessels / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A / Q000494:pharmacology
D020533:Angiogenesis Inhibitors; D043169:Endostatins; D042461:Vascular Endothelial Growth Factor A; D011092:Polyethylene Glycols