Modification of endostatin with polyethylene glycol and anti-angiogenesis evaluation on mice retina
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《中华医药杂志》英文版
[Abstract] Objective To modify endostatin (ES) with activated polyethylene glycol (PEG) and study the expression of vascular endothelial growth factor (VEGF) and pigment epithelium derived factor (PEDF) under the anti-angiogenesis effect of ES and PEG-ES on experimental retinal neovascularization (RNV). Methods The experimental RNV was induced by hyperbaric oxygen. The expression of new vessels and VEGF, PEDF protein were detected by hematoxylin and eosin staining and immunohistochemical study. Results ES was successfully modified with PEG blocked by cyclohexylamine in one end. The number of new vessels was significantly different between the two experimental groups and the control group. The expression of VEGF and PEDF protein was not different among three groups. Conclusions ES can be modified successfully with the method used in this study. ES and PEG-ES have remarkable effect on RNV, and the effect of PEG-ES is greater than that of the former. Considering that VEGF and PEDF protein expression is not different between the three groups, ES and PEG-ES may inhibit angiogenesis by interfering with the effecting processes of VEGF and PEDF, and not by depressing the expression of VEGF or improving expression of PEDF.
[Key words] endostatin;blood vessel,retinal;polyethylene glycol;pigment epithelium derived factor;vascular endothelial growth factor
INTRODUCTION Prevalence of diseases such as diabetic retinopathy, age-related macular degeneration and neovascular glaucoma is on the rise in developed and developing countries. These diseases are associated with intraocular neovascularization for which there is no effective cure at present. The therapeutic options in the management of these diseases include laser therapy, vitreous microsurgery, etc. These treatment options however do not resolve the intraocular neovascularization directly but indirectly by reducing retinal ischemia. Current focus in the management of intraocular neovascular diseases is using anti-angiogenic agents. In 1997, O'reilly of Harvard University extracted a protein that was 184 amino segment with 20kD in the C end of collagen ⅩⅧ, from the cell culture fluid of rat endothelium tumor[1]. This protein was named endostatin (ES). Experiments have proved that ES has selective inhibitive effect on endothelial cell proliferation, and has curative effect on experimental lung cancer, melanoma and fibrosarcoma.[2,3]. Clinical trials have shown that rational dose of ES for humans is 300mg/m2·d, and its half-life in human body is (10.7±4.1) h[4]. However, such a high dose may be substantially expensive and may also affect normal blood vessels; this being the most important obstacle to clinical application of ES. We can promote the clinical application of ES if its bioavailability can be improved and also extended its half-life. In this article, the authors describe the technique of using polyethylene glycol (PEG) to modify ES to enhance its half-life and improve its stability, and evaluate the effect of this modified ES on the retinal neovascularization (RNV) in mice.
MATERIALS AND METHODS
Reagents and Devices
PEG (MWt 10kD)(Biochemistry Research Institution, Pharmacy College, Shandong University),cicyclohexylcarbodiimide(DCC), N2 hydroxyl succinimide (NHS) (Zhongshan Inc.,Beijing,China), 2,2,6,6-tetramethyl-piperidinooxy (Zhongshan Inc., Beijing, China), cross-linked dextran gel (Sephacryl
1. Medicine College of Shandong University,Jinan 250012,Shandong Province,China
2. Pharmacy College of Shandong University,Jinan 250012,Shandong Province,China
3. Ji'nan Central Hospital,Jinan 250013,Shandong Province,China
Correspondence to LI Zhi-wei,Medicine college of Shandong University,Jinan 250012,Shandong Province,China
E-mail:lizhiweiii5@163.com-200)(Amersham Inc.), ES(Biochemistry Resear ching Institution, Pharmacy College, Shandong University), rabbit polyclonal antibody against mouse VEGF( Zhizhuang Inc. Shanghai, China), rabbit polyclonal antibody against mouse PEDF(Zhizhuang Inc. Shanghai, China), avidin-biotinylated-enzyme complex kits (ABC)(Boster Inc. Wuhan, China), ELISA kits (Zhizhuang Inc., Shanghai, China), calf serum(Sijiqing Inc. Hangzhou, China). Other reagents used included analytical reagent, chromatograph of gel permeation(GPC) (Waters Series: Pump 515, RI detector 2410, Column HT3-5-6)(Bruke Inc),infrared spectrometer(FITR vector 22)(Bruker Inc), oxygen analyser(XPO-318)( Deyu Inc., Beijing,China), hyper-oxygen box (Tong'an Medical Instrument Inc. Tong'an, China), computer image analysis system(KS 400 image analysis software package)(Media Cybernetic Inc.), 3 homochronous gestational C57 mice (the Animal Experiment Center of Shandong University)
METHODS
Preparation of Polyethylene Glycol Dioic Acid
1 mol/L PEG of was dissolved in dichlormethane, then 2,2,6,6-tetramethyl-piperidinooxy and microamount nitric acid were put into the liquor. The polyethylene glycol dioic acid(HOOCCH2-O-(CH2CH2O)2-CH2COOH)was precipitated as crystal form after 6～24 hours.
Preparation the Activated PEG Blocked by Cyclohexylamine in One End
5 g polyethylene glycol dioic acid was put into depurated dichlormethane, then double molal weight 2,2,6,6-tetramethyl-piperidinooxy 10 mol/L and 1.2 times molal weight cyclohexylamine were added, after 48 hours at room temperature, anhydrous diethyl ether was put into liquor to precipitate product. The product was dried in vacuum and determined by infrared spectrum.
Modifying ES with the Activated PEG Blocked with Cyclohexylamine in One End
1 g/L ES of 200 μl was dropped into 4 ependoff tubes (2ml) respectively and the pH of the liquor was regulated to the range between 6.0 and 8.0 with phosphate buffer, which was followed by adding the activated PEG into the mixed liquor. The proportion of protein to modifier should be around 1∶5. The reaction was ended with 2 mol/L aminoacetic acid after 30 min. The product was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
Abstraction and Purification of the Modified Product and Evaluation of Its Bioactivity
The modified ES was abstracted by Sephacryl-200. The bioactivity of ES and PEG-ES were evaluated by cell short term culture combined with H-TDR penetration method reported by Gillis[5].
Development of Experimental RNV
Three gestational C57 mice were bred, and 30 mice born of the 3mother mice were selected randomly when they were 7 days of age. These 30 new born mice and 3 mother mice were put in half airtight hyper-oxygen box. The concentration of oxygen was maintained at (75 ±2)% in the box and the concentration was measured 5 times per day. The room temperature was maintained at (23 ±2) ℃. After 5 days in the hyperbaric oxygen chamber, the new born mice were exposed to normal atmosphere for 7 days[6].
Anti-angiogenic Therapy
The 30 new born mice were divided randomly into 3 groups: group A as the control group, group B treated with ES(Table 1), group C treated with PEG-ES. Endostatin(1 g/L) or PEG ES (1 g/L) or normal saline 0.1 ml was injected into the vitreous cavities of mice on 12th day after birth. The mice were killed through intraperitoneal injecting excess 3 g/L pentobarbital and the eyeballs were extirpated as soon as possible and the lids were kept apart using lid speculum. The drug was injected into the vitreous cavity by inserting the 30 gauge needle 1mm behind the superotemporal or inferotemporal limbus. The needle entered the eye forming a 45° angle with the visual axis. After injection a sterilized cotton swab was pressed at the injection site to stop bleeding and antibiotic eyedrops was applied.
Table 1 General information of each group
The eyeballs were fixed in 4% polymethanal phosphoric acid buffer in 4℃ and embedded with paraffin,and cut into 6 microns thick sections，continuous sections through sagittal view in the sphere regarding the papilla optica as the center and 3 PD in radius(the papilla optica was obviated from slicing).
Staining and Immunohistochemistry
HE Stain Study Twenty sections were selected from each group and stained with hematoxylin eosin (HE) stain. The number of endothelial cell nuclei that have broken through the inner limiting membrane were counted under light microscope.
VEGF: Immunohistochemical Study
Twenty sections prepared as indicated in step Anti-angiogenic Therapy were selected from each group. The slides were treated with PBS diluted by 10% horse serum for 10 min at 37℃, then added with rabbit polyclonal antibody against mouse VEGF at 37℃ for 30 min and incubated at 4℃ overnight. After the incubation the slides were added with secondary antibody at 37℃ for 30 min. After that, the slices were treated with ABC reagent at room temperature for 30 min,which was followed by immersion in DAB coloring reagent at room temperature for 15 min. As a negative control, the incubation was performed with the phosphoric acid buffer instead of rabbit polyclonal antibody against mouse VEGF. Between each step, the slices were washed with PBS for 3 times.
PEDF: Immunohistochemical Study
Twenty sections were selected from each group. The slices were deparaffinized and treated with 3% H2O2 solution at 37 ℃ for 15 min, and immersed in EDTA solution at 95 ℃ for 10 min, then naturally cooled to room temperature. The slices were then blocked with goat serum blocking reagent at 37 ℃ for 15 min. After blocking, primary antibody, rabbit polyclonal antibody against mouse PEDF was applied to slices at 37℃ for 60 min and then at 4℃ overnight. After that, the slices were treated with secondary antibody marked with horseradish peroxidase at 37℃for 12 min.The slices were incubated with streptomycin avidin-biotin-peroxidase 37℃for 12 min. As chromogen, ABC reagent was used at room temperature for 5 min. At the last step, the hematoxylin was used to restain. Between each step, the slices were washed with PBS for 3 times. For control staining, PBS was used instead of primary antibody. The slices were observed under light microscope to detect the localization of immunoreactivity of PEDF.
Analysis of Photographs
The analysis was performed with KS 400 image analysis software package. The gray scale of VEGF and PEDF of each slide were measured and the average gray scale of each group was calculated. Then the results were analyzed by using a one-way analysis of variance (ANOVA) with SPSS 11.5 software package.
RESULTS
Estimation of Polyethylene Glycol Dioic Acid
The yield of polyethylene glycol dioic acid was measured by acid-base titration. Using 0.01mol/L NaOH as volumetric solution and phenolphthalein as indicator, we get the result that the yield is 99%.
Estimation of Activated PEG Blocked with Cyclohexylamine in One End
The PEG was used as negative control. KBr tabletting was applied to perform infrared spectrum determination. PEG has no absorption from 1 600 cm-1 to 1 800 cm-1 under infrared spectrum, however, the activated PEG has distinct absorption from 1 600 cm-1 to 1 700 cm-1 which demonstrates there has amide carbonyl group formed by cyclohexylamine and carboxyl group,and from 1 700 cm-1 to 1 800 cm-1 which demonstrates there has ester bond formed by NHS and carboxyl group.
Analysis of ES and PEG-ES by SDS-PAGE
The MWt of unmodified ES is around 20.4 kD, and that of ES modified with activated PEG which was not blocked with cyclohexylamine is around 31 kD. The MWt of ES modified with the activated PEG blocked with cyclohexylamine in one end is around 38 kD. However, at pH 8, there was only one redundant strip around 46kD on the electrophoretogram,which meaned the activating reaction reached the anticipated aim(Figure 1).
Figure 1 Electrophoretogram of the product mixture containing
ES and PEG-ES
Extraction of Modified Product and Evaluation of Its Bioactivity
The modified ES was abstracted by Sephacryl-200(1.0 cm×100 cm). The eluent is pH 7.0 phosphate buffer containing 0.15 mol/ L NaCl, and the eluting velocity was 0.5 ml/ min. After the abstraction, we got the unmodified ES, the ES combined with one PEG chain and the ES combined with two PEG chains from the product mixture. Through the evaluation of bioactivity and the measure of protein concentration, the specific activity of these three product were measured as 5. 72 ×106 IU/ mg,4. 02 ×106 IU/ mg and 1.68 ×106 IU/ mg respectively.
Effects of ES and PEG-ES on RNV
The endothelial cell of new vessels breaking through the inner limiting membrane could be observed in all the eyes studied(100%). Compared with group A,the nuclear count suggestive of neovascularization in group B and group C were reduced by 57.40%(P<0.01),41.96%(P<0.01)respectively(Table 2).
Table 2 Gray scale degree of VEGF and PEDF and the number of new vessels (±s)
Hematoxylin Eosin Stain
The average number of new vessels of each slice of group A,B,C was 251.23±22.25, 145.82±22.12 and 107.00±11.12 respectively. There was statistically significant differences among the 3 groups (P<0.01 ) (Figure 2).
Figure 2 Photograph of HE stain of RNV.A: Control group;B: Group treated with ES;C: Group treated with PEG-ES. The atrophy of new vessels and the decrease of their number can be observed from A to C
Immunohistochemistry Results
The location the of immunoreactivity of VEGF and PEDF were indicated by yellow brown and flavo-green particles in the kytoplasm respectively. There was no significant difference in the ratios of VEGF(Figure 3) and PEDF (P>0.05) (Figure4).
Figure 3 Immunohistochemistry stain of VEGF protein.A: Control group;B: Group treated with ES;C: Group treated with PEG-ES. Positive reaction was detected as yellow brown. The number of new vessels had decreased from A to C ,however ,the gray scale degree has not changed remarkable(P>0.05)
Figure 4 Immunohistochemistry stain of PEDF protein. A: Control group;B: Group treated with ES;C: Group treated with PEG-ES. Positive reaction was detected as flavo-green. The number of new vessels had decreased from A to C ,however ,the gray scale degree has not changed remarkable(P>0.05)
DISCUSSION
Endostatin, a proteolytic fragment of collagen XVⅢ, found in mouse angioendothelioma by O'Reilly in 1997[1], is a potent inhibitor of angiogenesis and suppresses neovascularization and tumour growth. However, the inhibitory mechanism of endostatin in human endothelial cells has not been characterized yet. Furthermore, clinical trials have shown that the half-life and bioavailability of simple ES is rather inadequate for the clinical therapy.
In this study, we modified ES with 3 chemical steps. Step 1: Polyethylene glycol dioic acid was produced from PEG, which provided 2 carboxyl groups for cyclohexylamine and NHS used in next step. Step 2: PEG was blocked by cyclohexylamine in one end, which prevented ES from excessive modification.Step 3: ES was modified by PEG prepared in step 2. The infrared spectrum measurement and SDS-PAGE all showed this method had blocked PEG and modified ES successfully.
The evaluation of the product's bioactivity shows that the bioactivity of ES modified with PEG in one end was better than that of simple ES and ES modified with two PEG chains in both ends. That is to say, the bioactivity of ES may decrease when it was modified excessively.
We also observed in these oxygen inducing RNV models that ES had remarkable effects on RNV and the changes of number of new vessels was correlated to the modification of ES. It is possible that some of the epitopes of ES were shielded by PEG, thereby prevent ES from the recognition and elimination of the immune system, thus prolong the bioavailability of ES. Moreover PEG increased the MWt of ES which might result in prolonging the half-life of ES and the biological distribution of ES was also changed.
In the retina, angiogenesis is regulated by 2 counterbalancing systems: angiogenic stimulators, such as vascular endothelial growth factor (VEGF), and angiogenic inhibitors, such as angiostatin and pigment epithelium derived factor (PEDF) [5~9]. Under pathological conditions, such as diabetic retinopathy and (ROP), regions of the retina become ischemic. Local ischemia increases the production of angiogenic stimulators and decreases the production of angiogenic inhibitors, breaking the balance between the positive and negative regulators of angiogenesis[10]. As a result, endothelial cells overproliferate, leading to retinal neovascularization[10,11].The image analysis displayed that there was no remarkable differences in the expression of VEGF and PEDF among group A,B,C, which suggests that ES might work by inhibiting the bioactivity of VEGF,PEDF or their receptors and not by inhibiting expression of VEGF or by improving expression of PEDF.
In summary, the excessive modification may decrease the bioactivity of ES,and the chemical modification used in this study may improve the efficacy of ES as an anti-angiogenic agent. Furthermore, the remarkable differences noted in retinal neovascularization indicates that PEG-ES has stronger effects on RNV induced by oxygen than simple ES. However the mechanism by which modified ES inhibits RNV needs further research.
Acknowledgements
We are thankful to Biochemistry Researching Institute of Shandong University. We also thank Zhizhuang, Inc. and Boster Inc. for providing primary and secondary antibody.We are indebted to our colleagues and our families for their continued support.
Funding:This work was supported by the Doctor Grant(2004042212)from the Ministry of Education,China
REFERENCES
1. Amano S, Kuroki M, Tolentino M, et al. Requirement for vascular endothelial growth factor in wound-related corneal neovascularization. Invest Ophthalmol Vis Sci,1998,39: 18-22.
2. Bocci G, Danesi R, Benelli U, et al. Inhibitory effect of suramin in rat models of angiogenesis in vitro and in vivo. Cancer Chemother Pharmacol,1999,43:205-212.
3. Murata T, Ishibashi T, Yoshikawa H, et al. Tecogalan sodium inhibits corneal neovascularization induced by basic fibroblast growth factor. Ophthalmic Res,1995,27:330-334.
4. Ziche, M, Donnini, S, Morbidelli, L, et al. Linomide blocks angiogenesis by breast carcinoma VEGF transfectants. Br. J. Cancer,1998,77:1123-1129.
5. Gills S, Ferm M M, Ward D U. T-cell growth factor: parameters of production and a quantitative microassay for activity. J Immunol,1978,120:2027.
6. Cao Hui, Hu Hong Hui, Xu Xun, Fan Ying, Wang Fang, Zhang Xi. Preparation of animal model of high oxygen inducing retinal neovascularizaton. Rec Adv Ophthalmol, 2003,23( 5):335-337.
7. Becerra SP. Structure-function studies on PEDF: a noninhibitory serpin with neurotrophic activity. Adv Exp Med Biol,1997,425:223-237.
8. Pierce EA, Avery RL, Foley ED, et al. Vascular endothelial growth factor/vascular permeability factor expression in a mouse model of retinal neovascularization. Proc Natl Acad Sci U S A,1995,92:905-909.
9. Miller JW, Adamis AP, Aiello LP. Vascular endothelial growth factor in ocular neovascularization and proliferative diabetic retinopathy. Diabetes Metab Rev,1997,13:37-50.
10. Wu YQ, Notario V, Chader GJ, Becerra SP. Identification of pigment epithelium-derived factor in the interphotoreceptor matrix of bovine eyes. Protein Expr Purif,1995,6:447-456.
11.Dawson DW, Volpert OV, Gillis P, Crawford SE, Xu H, Benedict W, Bouck NP.Pigment epithelium-derived factor: a potent inhibitor of angiogenesis. Science,1999,285:245-258.
(Editor Emilia)(LI Zhi-wei1, WANG Feng-sh)
[Key words] endostatin;blood vessel,retinal;polyethylene glycol;pigment epithelium derived factor;vascular endothelial growth factor
INTRODUCTION Prevalence of diseases such as diabetic retinopathy, age-related macular degeneration and neovascular glaucoma is on the rise in developed and developing countries. These diseases are associated with intraocular neovascularization for which there is no effective cure at present. The therapeutic options in the management of these diseases include laser therapy, vitreous microsurgery, etc. These treatment options however do not resolve the intraocular neovascularization directly but indirectly by reducing retinal ischemia. Current focus in the management of intraocular neovascular diseases is using anti-angiogenic agents. In 1997, O'reilly of Harvard University extracted a protein that was 184 amino segment with 20kD in the C end of collagen ⅩⅧ, from the cell culture fluid of rat endothelium tumor[1]. This protein was named endostatin (ES). Experiments have proved that ES has selective inhibitive effect on endothelial cell proliferation, and has curative effect on experimental lung cancer, melanoma and fibrosarcoma.[2,3]. Clinical trials have shown that rational dose of ES for humans is 300mg/m2·d, and its half-life in human body is (10.7±4.1) h[4]. However, such a high dose may be substantially expensive and may also affect normal blood vessels; this being the most important obstacle to clinical application of ES. We can promote the clinical application of ES if its bioavailability can be improved and also extended its half-life. In this article, the authors describe the technique of using polyethylene glycol (PEG) to modify ES to enhance its half-life and improve its stability, and evaluate the effect of this modified ES on the retinal neovascularization (RNV) in mice.
MATERIALS AND METHODS
Reagents and Devices
PEG (MWt 10kD)(Biochemistry Research Institution, Pharmacy College, Shandong University),cicyclohexylcarbodiimide(DCC), N2 hydroxyl succinimide (NHS) (Zhongshan Inc.,Beijing,China), 2,2,6,6-tetramethyl-piperidinooxy (Zhongshan Inc., Beijing, China), cross-linked dextran gel (Sephacryl
1. Medicine College of Shandong University,Jinan 250012,Shandong Province,China
2. Pharmacy College of Shandong University,Jinan 250012,Shandong Province,China
3. Ji'nan Central Hospital,Jinan 250013,Shandong Province,China
Correspondence to LI Zhi-wei,Medicine college of Shandong University,Jinan 250012,Shandong Province,China
E-mail:lizhiweiii5@163.com-200)(Amersham Inc.), ES(Biochemistry Resear ching Institution, Pharmacy College, Shandong University), rabbit polyclonal antibody against mouse VEGF( Zhizhuang Inc. Shanghai, China), rabbit polyclonal antibody against mouse PEDF(Zhizhuang Inc. Shanghai, China), avidin-biotinylated-enzyme complex kits (ABC)(Boster Inc. Wuhan, China), ELISA kits (Zhizhuang Inc., Shanghai, China), calf serum(Sijiqing Inc. Hangzhou, China). Other reagents used included analytical reagent, chromatograph of gel permeation(GPC) (Waters Series: Pump 515, RI detector 2410, Column HT3-5-6)(Bruke Inc),infrared spectrometer(FITR vector 22)(Bruker Inc), oxygen analyser(XPO-318)( Deyu Inc., Beijing,China), hyper-oxygen box (Tong'an Medical Instrument Inc. Tong'an, China), computer image analysis system(KS 400 image analysis software package)(Media Cybernetic Inc.), 3 homochronous gestational C57 mice (the Animal Experiment Center of Shandong University)
METHODS
Preparation of Polyethylene Glycol Dioic Acid
1 mol/L PEG of was dissolved in dichlormethane, then 2,2,6,6-tetramethyl-piperidinooxy and microamount nitric acid were put into the liquor. The polyethylene glycol dioic acid(HOOCCH2-O-(CH2CH2O)2-CH2COOH)was precipitated as crystal form after 6～24 hours.
Preparation the Activated PEG Blocked by Cyclohexylamine in One End
5 g polyethylene glycol dioic acid was put into depurated dichlormethane, then double molal weight 2,2,6,6-tetramethyl-piperidinooxy 10 mol/L and 1.2 times molal weight cyclohexylamine were added, after 48 hours at room temperature, anhydrous diethyl ether was put into liquor to precipitate product. The product was dried in vacuum and determined by infrared spectrum.
Modifying ES with the Activated PEG Blocked with Cyclohexylamine in One End
1 g/L ES of 200 μl was dropped into 4 ependoff tubes (2ml) respectively and the pH of the liquor was regulated to the range between 6.0 and 8.0 with phosphate buffer, which was followed by adding the activated PEG into the mixed liquor. The proportion of protein to modifier should be around 1∶5. The reaction was ended with 2 mol/L aminoacetic acid after 30 min. The product was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
Abstraction and Purification of the Modified Product and Evaluation of Its Bioactivity
The modified ES was abstracted by Sephacryl-200. The bioactivity of ES and PEG-ES were evaluated by cell short term culture combined with H-TDR penetration method reported by Gillis[5].
Development of Experimental RNV
Three gestational C57 mice were bred, and 30 mice born of the 3mother mice were selected randomly when they were 7 days of age. These 30 new born mice and 3 mother mice were put in half airtight hyper-oxygen box. The concentration of oxygen was maintained at (75 ±2)% in the box and the concentration was measured 5 times per day. The room temperature was maintained at (23 ±2) ℃. After 5 days in the hyperbaric oxygen chamber, the new born mice were exposed to normal atmosphere for 7 days[6].
Anti-angiogenic Therapy
The 30 new born mice were divided randomly into 3 groups: group A as the control group, group B treated with ES(Table 1), group C treated with PEG-ES. Endostatin(1 g/L) or PEG ES (1 g/L) or normal saline 0.1 ml was injected into the vitreous cavities of mice on 12th day after birth. The mice were killed through intraperitoneal injecting excess 3 g/L pentobarbital and the eyeballs were extirpated as soon as possible and the lids were kept apart using lid speculum. The drug was injected into the vitreous cavity by inserting the 30 gauge needle 1mm behind the superotemporal or inferotemporal limbus. The needle entered the eye forming a 45° angle with the visual axis. After injection a sterilized cotton swab was pressed at the injection site to stop bleeding and antibiotic eyedrops was applied.
Table 1 General information of each group
The eyeballs were fixed in 4% polymethanal phosphoric acid buffer in 4℃ and embedded with paraffin,and cut into 6 microns thick sections，continuous sections through sagittal view in the sphere regarding the papilla optica as the center and 3 PD in radius(the papilla optica was obviated from slicing).
Staining and Immunohistochemistry
HE Stain Study Twenty sections were selected from each group and stained with hematoxylin eosin (HE) stain. The number of endothelial cell nuclei that have broken through the inner limiting membrane were counted under light microscope.
VEGF: Immunohistochemical Study
Twenty sections prepared as indicated in step Anti-angiogenic Therapy were selected from each group. The slides were treated with PBS diluted by 10% horse serum for 10 min at 37℃, then added with rabbit polyclonal antibody against mouse VEGF at 37℃ for 30 min and incubated at 4℃ overnight. After the incubation the slides were added with secondary antibody at 37℃ for 30 min. After that, the slices were treated with ABC reagent at room temperature for 30 min,which was followed by immersion in DAB coloring reagent at room temperature for 15 min. As a negative control, the incubation was performed with the phosphoric acid buffer instead of rabbit polyclonal antibody against mouse VEGF. Between each step, the slices were washed with PBS for 3 times.
PEDF: Immunohistochemical Study
Twenty sections were selected from each group. The slices were deparaffinized and treated with 3% H2O2 solution at 37 ℃ for 15 min, and immersed in EDTA solution at 95 ℃ for 10 min, then naturally cooled to room temperature. The slices were then blocked with goat serum blocking reagent at 37 ℃ for 15 min. After blocking, primary antibody, rabbit polyclonal antibody against mouse PEDF was applied to slices at 37℃ for 60 min and then at 4℃ overnight. After that, the slices were treated with secondary antibody marked with horseradish peroxidase at 37℃for 12 min.The slices were incubated with streptomycin avidin-biotin-peroxidase 37℃for 12 min. As chromogen, ABC reagent was used at room temperature for 5 min. At the last step, the hematoxylin was used to restain. Between each step, the slices were washed with PBS for 3 times. For control staining, PBS was used instead of primary antibody. The slices were observed under light microscope to detect the localization of immunoreactivity of PEDF.
Analysis of Photographs
The analysis was performed with KS 400 image analysis software package. The gray scale of VEGF and PEDF of each slide were measured and the average gray scale of each group was calculated. Then the results were analyzed by using a one-way analysis of variance (ANOVA) with SPSS 11.5 software package.
RESULTS
Estimation of Polyethylene Glycol Dioic Acid
The yield of polyethylene glycol dioic acid was measured by acid-base titration. Using 0.01mol/L NaOH as volumetric solution and phenolphthalein as indicator, we get the result that the yield is 99%.
Estimation of Activated PEG Blocked with Cyclohexylamine in One End
The PEG was used as negative control. KBr tabletting was applied to perform infrared spectrum determination. PEG has no absorption from 1 600 cm-1 to 1 800 cm-1 under infrared spectrum, however, the activated PEG has distinct absorption from 1 600 cm-1 to 1 700 cm-1 which demonstrates there has amide carbonyl group formed by cyclohexylamine and carboxyl group,and from 1 700 cm-1 to 1 800 cm-1 which demonstrates there has ester bond formed by NHS and carboxyl group.
Analysis of ES and PEG-ES by SDS-PAGE
The MWt of unmodified ES is around 20.4 kD, and that of ES modified with activated PEG which was not blocked with cyclohexylamine is around 31 kD. The MWt of ES modified with the activated PEG blocked with cyclohexylamine in one end is around 38 kD. However, at pH 8, there was only one redundant strip around 46kD on the electrophoretogram,which meaned the activating reaction reached the anticipated aim(Figure 1).
Figure 1 Electrophoretogram of the product mixture containing
ES and PEG-ES
Extraction of Modified Product and Evaluation of Its Bioactivity
The modified ES was abstracted by Sephacryl-200(1.0 cm×100 cm). The eluent is pH 7.0 phosphate buffer containing 0.15 mol/ L NaCl, and the eluting velocity was 0.5 ml/ min. After the abstraction, we got the unmodified ES, the ES combined with one PEG chain and the ES combined with two PEG chains from the product mixture. Through the evaluation of bioactivity and the measure of protein concentration, the specific activity of these three product were measured as 5. 72 ×106 IU/ mg,4. 02 ×106 IU/ mg and 1.68 ×106 IU/ mg respectively.
Effects of ES and PEG-ES on RNV
The endothelial cell of new vessels breaking through the inner limiting membrane could be observed in all the eyes studied(100%). Compared with group A,the nuclear count suggestive of neovascularization in group B and group C were reduced by 57.40%(P<0.01),41.96%(P<0.01)respectively(Table 2).
Table 2 Gray scale degree of VEGF and PEDF and the number of new vessels (±s)
Hematoxylin Eosin Stain
The average number of new vessels of each slice of group A,B,C was 251.23±22.25, 145.82±22.12 and 107.00±11.12 respectively. There was statistically significant differences among the 3 groups (P<0.01 ) (Figure 2).
Figure 2 Photograph of HE stain of RNV.A: Control group;B: Group treated with ES;C: Group treated with PEG-ES. The atrophy of new vessels and the decrease of their number can be observed from A to C
Immunohistochemistry Results
The location the of immunoreactivity of VEGF and PEDF were indicated by yellow brown and flavo-green particles in the kytoplasm respectively. There was no significant difference in the ratios of VEGF(Figure 3) and PEDF (P>0.05) (Figure4).
Figure 3 Immunohistochemistry stain of VEGF protein.A: Control group;B: Group treated with ES;C: Group treated with PEG-ES. Positive reaction was detected as yellow brown. The number of new vessels had decreased from A to C ,however ,the gray scale degree has not changed remarkable(P>0.05)
Figure 4 Immunohistochemistry stain of PEDF protein. A: Control group;B: Group treated with ES;C: Group treated with PEG-ES. Positive reaction was detected as flavo-green. The number of new vessels had decreased from A to C ,however ,the gray scale degree has not changed remarkable(P>0.05)
DISCUSSION
Endostatin, a proteolytic fragment of collagen XVⅢ, found in mouse angioendothelioma by O'Reilly in 1997[1], is a potent inhibitor of angiogenesis and suppresses neovascularization and tumour growth. However, the inhibitory mechanism of endostatin in human endothelial cells has not been characterized yet. Furthermore, clinical trials have shown that the half-life and bioavailability of simple ES is rather inadequate for the clinical therapy.
In this study, we modified ES with 3 chemical steps. Step 1: Polyethylene glycol dioic acid was produced from PEG, which provided 2 carboxyl groups for cyclohexylamine and NHS used in next step. Step 2: PEG was blocked by cyclohexylamine in one end, which prevented ES from excessive modification.Step 3: ES was modified by PEG prepared in step 2. The infrared spectrum measurement and SDS-PAGE all showed this method had blocked PEG and modified ES successfully.
The evaluation of the product's bioactivity shows that the bioactivity of ES modified with PEG in one end was better than that of simple ES and ES modified with two PEG chains in both ends. That is to say, the bioactivity of ES may decrease when it was modified excessively.
We also observed in these oxygen inducing RNV models that ES had remarkable effects on RNV and the changes of number of new vessels was correlated to the modification of ES. It is possible that some of the epitopes of ES were shielded by PEG, thereby prevent ES from the recognition and elimination of the immune system, thus prolong the bioavailability of ES. Moreover PEG increased the MWt of ES which might result in prolonging the half-life of ES and the biological distribution of ES was also changed.
In the retina, angiogenesis is regulated by 2 counterbalancing systems: angiogenic stimulators, such as vascular endothelial growth factor (VEGF), and angiogenic inhibitors, such as angiostatin and pigment epithelium derived factor (PEDF) [5~9]. Under pathological conditions, such as diabetic retinopathy and (ROP), regions of the retina become ischemic. Local ischemia increases the production of angiogenic stimulators and decreases the production of angiogenic inhibitors, breaking the balance between the positive and negative regulators of angiogenesis[10]. As a result, endothelial cells overproliferate, leading to retinal neovascularization[10,11].The image analysis displayed that there was no remarkable differences in the expression of VEGF and PEDF among group A,B,C, which suggests that ES might work by inhibiting the bioactivity of VEGF,PEDF or their receptors and not by inhibiting expression of VEGF or by improving expression of PEDF.
In summary, the excessive modification may decrease the bioactivity of ES,and the chemical modification used in this study may improve the efficacy of ES as an anti-angiogenic agent. Furthermore, the remarkable differences noted in retinal neovascularization indicates that PEG-ES has stronger effects on RNV induced by oxygen than simple ES. However the mechanism by which modified ES inhibits RNV needs further research.
Acknowledgements
We are thankful to Biochemistry Researching Institute of Shandong University. We also thank Zhizhuang, Inc. and Boster Inc. for providing primary and secondary antibody.We are indebted to our colleagues and our families for their continued support.
Funding:This work was supported by the Doctor Grant(2004042212)from the Ministry of Education,China
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(Editor Emilia)(LI Zhi-wei1, WANG Feng-sh)