Comparison of the antiangiogenic activity of modified RGDRGD-endostatin to endostatin delivered by gene transfer in vivo rabbit neovascularization model

Abstract

Purpose: Endostatin plays an important role in inhibiting corneal neovascularization (CNV). The aim of this study was to evaluate the antiangiogenic activities of lipid-mediated subconjunctival injection of the modified RGDRGD (arginine- glycin- aspartic- arginine- glycin- aspartic- endostatin gene in a rabbit model of neovascularization in vivo.

Methods: A modified human endostatin gene containing an RGDRGD motif was obtained by rapid site-directed mutagenesis. Forty New Zealand white rabbits underwent alkaline burn and developed CNV, which were randomly divided into four groups: an experimental control group, a PCI empty vector group, a PCI-endostatin group, and a PCI-RGDRGD-endostatin group. The vector, endostatin, and RGDRGD-endostatin groups received injections into the superior bulbar conjunctiva after the burn. An injection of 5 μg was given twice at 1-week intervals. Four eyes of two rabbits received neither treatment nor alkaline burn and served as absolute normal controls. The areas of CNV were monitored after 7 and 14 days. Corneas were examined by histology, and VEGF (vascular endothelial growth factor) and CD31 (platelet endothelial cell adhesion molecule-1) expression was detected by immunohistochemistry after 7 and 14 days. Retina, liver, and kidney were examined by histology, and CD38 expression in the inflammatory cells was detected by immunohistochemistry at 90 days.

Results: Subconjunctival injection of both native endostatin and modified RGDRGD-endostatin genes resulted in a significant suppression of CNV in vivo, with modified RGDRGD-endostatin being more effective than native endostatin. The mean concentration of VEGF in the PCI-RGDRGD-endostatin group significantly decreased compared to the means in the other groups. Upon histological examination, the endostatin-treated and RGDRGD-endostatin-treated eyes showed significantly less neovascular area and fewer vessels than the control and vector-injected groups. Retinal, hepatic, and renal tissue sections were normal, and there was no inflammatory cell infiltration observed.

Conclusions: Native and modified endostatin can significantly inhibit CNV by suppressing the expression of VEGF. However, modified endostatin with the RGDRGD motif is far more effective than the endostatin gene in antiangiogenic activity.

Figure 1

Construction of vectors containing human native and modified endostatin genes. Identification of PCI-endostatin and pCI-RGDRGD-endostatin. Lane 1: 15,000 marker; Lane 2: 2,000 marker; Lane 3: PCI-endostatin; Lane 4: PCI-endostatin (SacI +SalI); Lane 5: PCI-RGDRGD-endostatin; Lane 6: PCI-RGDRGD-endostatin (SacI +SalI); Lane 7: PCR product of native endostatin gene; Lane 8: PCR product of modified RGDRGD-endostatin gene.

Figure 2

The sequence analysis of the endostatin and modified RGDRGD-endostatin genes. A: The sequence analysis of the endostatin gene in the range of 70 to 100 bp. B: The sequence analysis of the modified RGDRGD-endostatin gene in the range of 70 to 97 bp. The sequence analysis indicated that a modified endostatin gene was obtained by delete alanine ala (A) and isoleucine ile (I) changed to asparticacid asp (D), and that the RGDRGD motif was successfully constructed.

Figure 3

RT–PCR analyses assessed the duration of endostatin or RGDRGD-endostatin mRNA expression in the rabbit cornea. The expression of the human endostatin or RGDRGD-endostatin gene (550 bp) was observed until 5 days after single subconjunctival gene injection, while there was no expression of after vector (V) injection. The results of rabbit GAPDH (465 bp) confirmed the relative amounts and fidelity of the total RNA samples.

Figure 4

Biomicroscopic findings in corneas after subconjunctival injection of endostatin-RGDRGD or endostatin or vector, and in untreated control corneas. Representative biomicroscopic findings of rabbit corneas are shown. The corneas were examined at 1, 7, and 14 days after injection. The endostatin-RGDRGD-treated eyes showed significantly less neovascular growth than endostatin-treated eyes or vector-treated eyes or untreated eyes at day 7, and peaked on day 14.

Figure 5

The inhibitory effect of modified RGDRGD-endostatin gene on the area of CNV and the expression VEGF in aqueous humor. A: The inhibitory effect of modified RGDRGD-endostatin gene on the area of CNV. The area of CNV was calculated by the number of newly developed vessels multiplied by the length of the vessels from the limbus, 7 and 14 days after the alkaline burn. B: The concentration of VEGF in aqueous humor (n=10, M±SD). The modified RGDRGD-endostatin gene has a significantly different effect compared with the native gene at 7 and 14 days. Data are expressed as a mean±SD, as well as at other days. ***p<0.001; **p<0.01; *p<0.05, when CNV with treated groups was compared with control group. ^tp<0.05, ^ttp<0.01, when CNV with the combination of RGDRGD-endostatin was compared with CNV treated with native endostatin.

Figure 6

Histologic profiles of the corneal buttons . Corneal buttons were collected on day 14 (A-E). Corneal button sections of normal (A),control (B), Vector (C), endostatin (D), and RGDRGD-endostatin (E) groups were stained with hematoxylin and eosin. Expression of VEGF in the corneal buttons was detected by immunohistochemistry (F-J). High levels of VEGF were detected in the control and vector groups, respectively (G-H); by contrast, only minute levels were detected in the endostatin and RGDRGD-endostatin gene groups (I-J), respectively . Light micrographs of CD31-stained corneas in an untreated eye after corneal denudation and the effects of RGDRGD-endostatin gene delivery on experimental corneal angiogenesis on day 14. Representative light micrographs of CD31-stained corneas given normal, control,vector,endostatin,or the RGDRGD-endostatin expression cassette are shown (each group; n=3; K-O).The absolute normal control group showed no neovascularization, the experimental normal and vector groups showed neovascular formation (arrowheads), the endostatin group showed less neovascular formation,while the RGDRGD-endostatin group showed significantly less vessels and smaller areas of neovascularization compared with endostatin-treated corneas.

Figure 7

Histological profiles of retinal, hepatic, and renal tissue sections at 90 days. Retinal, hepatic, and renal tissue sections were normal, and no inflammatory cell infiltration was observed (A-C). Immunohistochemical studies indicated that CD38+ cells were not detected in the retinal, hepatic, and renal tissue sections (E-G), respectively. In positive controls, the expression level of CD38 rised significantly. CD38+ cells (arrowheads) were detected in the hepatic tissue sections (D and H), respectively.