Acidic/neutral amino acid residues substitution in NH2 terminal of plasminogen kringle 5 exerts enhanced effects on corneal neovascularization.
OBJECTIVE
Recent results showed that plasminogen kringle 5 (K5) has improved inhibitory effect on human umbilical vein endothelial cells (HUVECs) viability when 5 acidic amino acids in NH2 terminal outside kringle domain were replaced by 5 serine residues (mutant K5, mK5). This study was designed to identify the enhanced antiangiogenic activity of mK5 in corneal neovascularization (CNV).


METHODS
Alkali burn-induced CNV was induced and treated with K5 and mK5 for 11 days. CNV and inflammation were evaluated by the CNV area and the inflammatory index, respectively. At the end of treatment, the corneas were removed for terminal deoxynucleotidyl transferase dUTP nick end labeling detection and immunohistochemistry. The effects of mK5 and K5 on HUVECs apoptosis were tested by MTT, BrdU, and flow cytometry. The expression levels of pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor (VEGF) were detected by Western blot.


RESULTS
In a rat model of CNV induced by alkali, topical treatment with mK5 significantly decreased the neovascular area and inflammation compared with the wild-type K5-treated group. Meanwhile, mK5 and K5 specifically inhibited the HUVECs proliferation and induced vascular endothelial cell apoptosis in vitro and in vivo, and mK5 exerted higher apoptosis induction. Toward the mechanism of action, both mK5 and K5 significantly upregulated the expression of PEDF and mildly downregulated the expression of VEGF. The elevation of PEDF/VEGF ratio induced by mK5 was higher than that by K5.


CONCLUSIONS
These findings suggest that mK5 has more effective therapeutic potential in CNV than wild-type K5.
Department of Biochemistry, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China.
Cen
C
Li
Lei
L
Li
Rui
R
Cheng
Zhiyu
Z
Dai
Chaoyang
C
Li
Yachao
Y
Yao
Ti
T
Zhou
Zhonghan
Z
Yang
Guoquan
G
Gao
Xia
X
Yang
D019943:Amino Acid Substitution / Q000235:genetics*; D024342:Amino Acids, Acidic; D021542:Amino Acids, Neutral; D020533:Angiogenesis Inhibitors / Q000627:therapeutic use*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D016510:Corneal Neovascularization / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D004195:Disease Models, Animal; D004730:Endothelium, Vascular / Q000473:pathology; D005136:Eye Proteins / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry; D061307:Human Umbilical Vein Endothelial Cells / Q000473:pathology; D006801:Humans; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D016297:Mutagenesis, Site-Directed; D009414:Nerve Growth Factors / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000627:therapeutic use*; D010958:Plasminogen / Q000235:genetics / Q000627:therapeutic use*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015843:Serpins / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D000099174:Pigment Epithelium-Derived Factor
D024342:Amino Acids, Acidic; D021542:Amino Acids, Neutral; D020533:Angiogenesis Inhibitors; D005136:Eye Proteins; D009414:Nerve Growth Factors; D010446:Peptide Fragments; D010958:Plasminogen; D015843:Serpins; D042461:Vascular Endothelial Growth Factor A; D000099174:Pigment Epithelium-Derived Factor; C491499:plasminogen kringle 5; C467498:vascular endothelial growth factor A, rat