Effect of angiostatin to corneal epithelial cells, fibroblast and vascular endothelial cells in vitro
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《中华医药杂志》英文版
Effect of angiostatin to corneal epithelial cells, fibroblast and vascular endothelial cells in vitro(pdf)
1 Department of Ophthalmology, No. 421 Hospital of PLA, Guangzhou, Guangdong Province 510318, China
2 Anatomy Institute of Nanfang Medical University, Guangzhou, Guangdong Province 510515, China
Correspondence to LIU Hai-jun,MD.,Department of Ophthalmology, No. 421 Hospital of PLA, Guangzhou, Guangdong Province 510318, China
Tel:+86-20-61636610, E-mail:TOP328@163.net
[Abstract] Objective To investigate the inhibited effect of angiostatin (AS) to corneal epithelial cell, fibloblasts and the vascular endothelial cells. Methods The experimental studies of cells culture in vitro: After corneal epithelial cells, fibroblasts and the vascular endothelial cells were separated, all these cells were cultured and transferred in the incubation(37 ℃, 5%CO2). When transferred for the second generation, cells were inoculated in different culture pore. Each kind of cells were transferred in 96 wells to culture respectively, and were divided into four groups: A,the control group, B, C,D AS groups; each group had 24 wells. When cells anchoring, 24 wells of A group were added with 200 μl DMEM culture medium, B,C,D groups were added with 0.1, 0.3,0.5 μg/ml AS-DMEM culture medium, respectively. The conditions of cellular growth were observed in 24 and 48 hours, and the OD values of all wells were examined by MTT means. This experiment was repeated for three times.Results (1) The inhibitory effects of AS to the corneal epithelial cells: In 24 h and 48 h after applied drugs, all the AS-groups were not significantly different compared with the control group (P>0.05). (2) The inhibitory effect of AS to the corneal fibroblast cells. At 24 h, there were some inhibitory effect in applied AS groups. At 48 h after applied AS, there were not markedly different between all groups applied with AS and the control group. (3) The inhibitory effects of AS to the vascular endothelial cells: The values of all groups applied with AS were not different compared to the control group at 24 h (P>0.05). In 48 hours, the OD values of these four groups were 0.557±0.123, 0.421±0.078, 0.390±0.031 and 0.386±0.262, respectively. The cellular inhibitory rates of B, C, D groups were 27.0%, 32.3% and 33.1%, there were significantly lower at 48 h (P<0. 05) compared with the control groups.Conclusion Angiostatin has no effect to the proliferation of corneal fibroblast cells and epithelial cells, but has partial inhibitory effects to the vascular endothelial cells, the effect will enhance with the concentration going up, but it did not inhibit the proliferation completely.
[Key words] angiostatin; corneal epithelial cells; fibroblast; vascular endothelial cells
INTRODUCTION
Angiostatin(AS) is one of the most powerful angiogenesis inhibitor, applying on local ocular is in experimental research stage. Corneal alkali burns is one of the various corneal injuries which can cause corneal neovascularization and blindness without effective drugs to treat it. It would be an important subject of ophthalmologist on how to control corneal neovascularization. There is not reports on study of inhibiting proliferation of corneal epithelial cells, corneal fibroblasts and vascular endothelial cells in vitro in China. This study was to explore inhibitory effect of AS on proliferation of vascular endothelial cells without affecting the corneal epithelial cells and fibroblasts.
MATERIALS AND METHODS
Materials
Drugs and reagents Recombinant human angiostatin (College of Life Sciences at Zhongshan University,Guangzhou,China)，streptomycin injection，penicillium injection，complete DMEM solution，10%fetal calf serum(FCS)，0.25%trypsin，Trypan blue，Hanks solution，DMSO analytically pure，MTT solution.
Donor Two 1-month-old New Zealand white rabbits, healthy and no eye diseases.
Instruments Stereomicroscope, inverted microscope, CO2 incubator,enzyme labelled instrument，96-well plate, 20 μl,100 μl,1 ml micro sample gun，microplate oscillator, blood cell counter.
Drug Preparation
Recombinant human angiostatin+ DMEM culture solution, concentrations: 0.1,0.3,0.5 μg/ml AS DMEM solution.
Isolation, culture and passage of corneal epithelial cells, keratocytes and vascular endothelial cells
Isolation, culture and passage of corneal epithelial cells De-epithelialized cornea was digested with 0.25% trypsin at 37 ℃ for 12 h， DMEM medium with 10%FCS was used to end digestion, scraped tissue under microscope stirring，blending,700 rpm/s， 5 min，supernatant removed, inoculated in the culture bottles, at 37 ℃,5%CO2, cultured in warm incubator, change solution for every three day.
Fibroblasts isolated, cultured and passaged Cutting stroma lamella by l mm piece, inoculated in dish, supplemented DMEM media with 10%FCS 1.5 ml cultured in warm incubator at 37 ℃,5%CO2, change 2/3 solution every day.
Isolation and passage of vascular endothelial cells Under aseptic conditions，take aorta of 1-month-old rabbit (length 20 cm)， squeeze out hematocele, set in preservative fluid (0.14 M,NaCl 0.04 M,KCl,0.001 M phosphate buffer，0.001 M glucose，pH 7.4，streptomycin 100 μ/ml,penicillin 100 μ/ml)at 4 ℃. Washing with Hanks solution to no blood in artery.Hemostatic forceps clamping one end of aorta， infusing 0.1% trypsin at other end， loosing hemostatic forceps after filled，washing lumen of aorta with Hanks solution 2 times.Then clamping one end of aorta with hemostatic forceps，infusing 0.25% trypsin at other end and clamped，bathing in beaker at 37 ℃, incubating 10 min，add cell enzyme solution in beaker，washing aorta with Hanks solution，washing fluid pouring in beaker，add FCS immediately，ending action of trypsin， centrifuged for 10 min，removing supernatant，supplementing DMEM solution (15% FCS，L- glutamate 2 mmol)，mixed completely， 0.5% Trypan blue staining，counting percent of viable cells，adjusting cell number at concentration of 1×105/ml in flask，change solution every 2 days，passage and culture after growth and fusion of the cells.Remove culture solution，add 0.125%trypsin，digest and isolate， removing supernatant，add DMEM solution to produce cell suspension，passage cells to new flask by ratio of 1∶2 for culturing. Conduct experiment after passage to second generation
Effects of Different Concentration of Angiostatin on Corneal Epithelial Cells
Fibroblasts and vascular endothelial cells inoculate second generation of corneal epithelial cells in 96 well plate at concentration of 1×104/ml，divided into 4 groups，A: control group 24 well，B,C and D as study group each 24 well. Add 0.1,0.3,0.5 μg/ml AS-DMEM in B,C and D respectively after cells anchoring，at 37 ℃,5%CO2 in incubator. The changes of morphology and amount of cells after 24,48 h were observed. MTT was used to determine OD，to evaluate proliferation and inhibition of the cells.
Corneal fibroblasts and vascular endothelial cells were treated with same methods.
MTT Colorimetric Assay
Principle Dehydrogenase in viable cells can reduct tetrazolium salt into insoluble blue material - formazan,deposit in cells. The phenomenon do not occur in dead cells. DMSO can dissolve blue crystal deposited in the cell.Color depth of the solution proportional to amount of formazan.Enzyme labelled instrument was used to determine OD,to count inhibitory rate.
Inhibition rate of the cells =[(control group OD -study group OD)/control group OD]×100%。
Method 4 h before colorimetry，add MTT solution(20 μl/well)；Suction of culture solution, and then add DMSO solution (150 μl/well), at room temperature, oscillate plate on oscillator for 10 min,crystal dissolved, determine each well OD (λ=570 nm)
Statistical Analysis
Statistical software SPSS10.0,One-Way ANOVA.
RESULTS
Inhibitory effects of AS on proliferation of corneal epithelial cell, fibroblasts and vascular endothelial cell (See Table 1，2，3).
Table 1 Inhibitory Effects of AS on Proliferation of Corneal Epithelial Cells
#P>0.05，△non inhibition
Table 2 Inhibitory Effects of AS on Proliferation of Corneal Fibroblasts
*P<0.05，#P>0.05
Table 3 Inhibitory Effects of AS on Proliferation of Vascular Endothelial Cells
#P>0.05，*P<0.05
Corneal epithelial cells and vascular endothelial cells were not inhibited after AS administered 24 h, however corneal filrablasts was inhibited relatively.48 h later, results was same as 24 h, but proliferation of vascular endothelial cell was not inhibited completely. Compared to control group, viable cells were decreased markedly.
DISCUSSION
Role of Angiostatin in Inhibition of Angiogenesis
AS is one of the most powerful angiogenesis inhibitor, it can inhibit tumor angiogenesis and migration[1,2], can obtained by plasminogen degradation with MMP-2,7,9[3]. Kim, et al. found that Klinglel-3 can inhibit bFGF or VEGF induced corneal neovascularization with dose-dependent mode. Kringlel-3 can decease white blood cell significantly in cornea in study group more than that in control group.Stack MS，et al. reported that AS was one of non-competed inhibitor[3], AS can inhibit stromal increased activity of plasminogen in microcirculation of the cell result in decreasing invasive activity of the cell. It is suggests that AS mediated regulation of plasminogen formation can influence cell migration and invasive capacity. Walter，et al. confirmed that AS can inhibit proliferation of vascular endothelial cell in vitro but plasminogen cannot do that[6].So it is suggest that there are specified AS receptor in vascular endothelial cell. AS is bound with plasminogen receptor on smooth muscle, excessive plasminogen and Epsilon - aminocaproic acid (EACA) can inhibit the binding.
AS bound with plasminogen receptor can block binding of plasminogen and plasminogen activator, and block activator formation later[7]. Smooth muscle cells participating vascular forming was limited as long as lack plasminogen activator around cells[8].So, vascular epithelial cells in inhibiting vascular forming is play the roles as inhibiting proliferation and migration of vascular epithelial cells and smooth muscle cells. Moser, et al. found that AS bound with subunitα/β of ATP synthetase to down-regulate proliferation and migration of endothelial cells to mediate effects of anti-angiogenesis[9].
So, AS can inhibit neovascularization mediated by angiogenesis factor bFGF and VEGF. Gabison F, et al. confirmed that plasminogen and angiostatin are produced in the cornea. They may play a role in preventing neovascularization and may contribute to the maintenance of corneal avascularity after excimer laser keratectomy[10]. Warejcka DJ, et al. suggested that conversion of plasminogen to angiostatin is cell-type dependent. Because corneal cells generate angiostatins, use of human angiostatins may be a mean of treating abnormal corneal neovascularization without the risks of side effects[11].
Experimental Results Analysis
AS 0.1, 0.3, 0.5 μg/ ml, after 24 h without significant inhibition effects on vascular endothelial cells, after 48 h inhibition effects were occurred, viable cells were decreased markedly. But compared with same group at 24 h, viable cells were increased markedly too. It is suggested that proliferation of vascular epithelial cells was not inhibited completely.With increasing of concentration of AS, inhibitory effects were increased slightly.AS is not inhibit synthesizing DNA of vascular epithelial cells, but it is compete specific targets on the cell surface with proliferation factors to inhibit proliferation of the cells. It has inhibitory effects but do not inhibit completely without growth factors in vitro. In this study, AS 0.1,0.3 and 0.5 μg/ml cannot inhibit proliferation of corneal epithelial cells significantly.Inhibitory effects after 24 h on fibroblasts may be caused by measurement error, it is unclear on the mechanism of that effects because we are not find any inhibitory effects after 48 h.
CONCLUSION
In vitro study showed that AS 0.1,0.3,0.5μg/ml can inhibit proliferation of vascular endothelial cells slightly in the absence of growth factor. Inhibitory effects increased with increasing of concentration of AS, but never inhibit completely. And non inhibitory effects were found in corneal epithelial cells and fibroblasts. We concluded that AS may compete specific targets on the cell surface with proliferation factors to inhibit proliferation of the cells.
REFERENCES
1. O'Reilly MS, Holmgren L, Shing Y, et al. Angiostatin:a circulating endothelial cell inhibitor that suppress angiogenesis and tumor growth. Cold-Spring-Harb-Symp-Quant-Boil, 1994, 59: 471-482.
2. O'Reilly MS, Holmgren L, Chen C, et al. Angiostatin induces and sustains dormancy of human primary tumors in mice. Nat Medicine, 1996, 2: 689-692.
3. O'Reilly MS, Wiederschain D, Stetler-Stevenson WG, et al. Regulatior of angiostatin production by matrix metalloproteinase-2 in a model of concomitant resistance. J Bio Chem, 1999,274: 19568-29571.
4. Kim JH, Kim JC. Shin SH, et al. The inhibitory effects of recombinant plasogen kringlel-3 on the neovascularization of rabbit cornea induced by angiogenin, bFGF and VEGF. Exp Mol Med, 1999, 31(4): 203-209.
5. Stack MS, Gately S, Bafetti LM, et al. Angiostatin inhibits endothelial andmelanoma celhlar invasion by bloking matrix-enhanced plasogen activation. Biochem J, 1999, 340(ptl): 77-84.
6. Walter JJ, Sane DC. Angiostatin binds to smooth muscle cells in the coronary artery and inhibits smooth musc〗e cell proliferation and migration in vitro. Arterioscler Thromb Vase Biol, 1999, 19: 2041-2048.
7. Hajjar KA, Harpel PC, Jaffe EA, et al. Bindingjof plasogen to cultured human endothelial cells. JBiol Chem, 1986, 261: 11656-11662.
8. Jackson CL, Raines EW, Ross R, et al. Role of endogenous platelet-derived growth factor in arterial smooth muscle cell migration after balloon catheter injury. Arterioscler Thromb, 1993, 13: 1218-1226.
9. Moser TL, Stack MS, Asplin I, et al. Angiostatin binds ATP synthase on the surface of human endothelial cells. Proc Natl Acad Sci USA, 1999, 96(6): 2811-2816.
10. Gabison F, Chang JH, Hernandez-Quintela E, et al. Anti-angiogenic role of angiostatin during corneal wound healing. Exp Eye Res, 2004,78(3):579-589.
11. Warejcka DJ, Vauhan KA, Bernstein AM, et al. Differential conversion of plasminogen to angiostatin by human corneal cell population. Mol VIS, 2005,11:859-868.
(Editor Jaque)(LIU Hai-jun1, ZHONG Shi-z)
1 Department of Ophthalmology, No. 421 Hospital of PLA, Guangzhou, Guangdong Province 510318, China
2 Anatomy Institute of Nanfang Medical University, Guangzhou, Guangdong Province 510515, China
Correspondence to LIU Hai-jun,MD.,Department of Ophthalmology, No. 421 Hospital of PLA, Guangzhou, Guangdong Province 510318, China
Tel:+86-20-61636610, E-mail:TOP328@163.net
[Abstract] Objective To investigate the inhibited effect of angiostatin (AS) to corneal epithelial cell, fibloblasts and the vascular endothelial cells. Methods The experimental studies of cells culture in vitro: After corneal epithelial cells, fibroblasts and the vascular endothelial cells were separated, all these cells were cultured and transferred in the incubation(37 ℃, 5%CO2). When transferred for the second generation, cells were inoculated in different culture pore. Each kind of cells were transferred in 96 wells to culture respectively, and were divided into four groups: A,the control group, B, C,D AS groups; each group had 24 wells. When cells anchoring, 24 wells of A group were added with 200 μl DMEM culture medium, B,C,D groups were added with 0.1, 0.3,0.5 μg/ml AS-DMEM culture medium, respectively. The conditions of cellular growth were observed in 24 and 48 hours, and the OD values of all wells were examined by MTT means. This experiment was repeated for three times.Results (1) The inhibitory effects of AS to the corneal epithelial cells: In 24 h and 48 h after applied drugs, all the AS-groups were not significantly different compared with the control group (P>0.05). (2) The inhibitory effect of AS to the corneal fibroblast cells. At 24 h, there were some inhibitory effect in applied AS groups. At 48 h after applied AS, there were not markedly different between all groups applied with AS and the control group. (3) The inhibitory effects of AS to the vascular endothelial cells: The values of all groups applied with AS were not different compared to the control group at 24 h (P>0.05). In 48 hours, the OD values of these four groups were 0.557±0.123, 0.421±0.078, 0.390±0.031 and 0.386±0.262, respectively. The cellular inhibitory rates of B, C, D groups were 27.0%, 32.3% and 33.1%, there were significantly lower at 48 h (P<0. 05) compared with the control groups.Conclusion Angiostatin has no effect to the proliferation of corneal fibroblast cells and epithelial cells, but has partial inhibitory effects to the vascular endothelial cells, the effect will enhance with the concentration going up, but it did not inhibit the proliferation completely.
[Key words] angiostatin; corneal epithelial cells; fibroblast; vascular endothelial cells
INTRODUCTION
Angiostatin(AS) is one of the most powerful angiogenesis inhibitor, applying on local ocular is in experimental research stage. Corneal alkali burns is one of the various corneal injuries which can cause corneal neovascularization and blindness without effective drugs to treat it. It would be an important subject of ophthalmologist on how to control corneal neovascularization. There is not reports on study of inhibiting proliferation of corneal epithelial cells, corneal fibroblasts and vascular endothelial cells in vitro in China. This study was to explore inhibitory effect of AS on proliferation of vascular endothelial cells without affecting the corneal epithelial cells and fibroblasts.
MATERIALS AND METHODS
Materials
Drugs and reagents Recombinant human angiostatin (College of Life Sciences at Zhongshan University,Guangzhou,China)，streptomycin injection，penicillium injection，complete DMEM solution，10%fetal calf serum(FCS)，0.25%trypsin，Trypan blue，Hanks solution，DMSO analytically pure，MTT solution.
Donor Two 1-month-old New Zealand white rabbits, healthy and no eye diseases.
Instruments Stereomicroscope, inverted microscope, CO2 incubator,enzyme labelled instrument，96-well plate, 20 μl,100 μl,1 ml micro sample gun，microplate oscillator, blood cell counter.
Drug Preparation
Recombinant human angiostatin+ DMEM culture solution, concentrations: 0.1,0.3,0.5 μg/ml AS DMEM solution.
Isolation, culture and passage of corneal epithelial cells, keratocytes and vascular endothelial cells
Isolation, culture and passage of corneal epithelial cells De-epithelialized cornea was digested with 0.25% trypsin at 37 ℃ for 12 h， DMEM medium with 10%FCS was used to end digestion, scraped tissue under microscope stirring，blending,700 rpm/s， 5 min，supernatant removed, inoculated in the culture bottles, at 37 ℃,5%CO2, cultured in warm incubator, change solution for every three day.
Fibroblasts isolated, cultured and passaged Cutting stroma lamella by l mm piece, inoculated in dish, supplemented DMEM media with 10%FCS 1.5 ml cultured in warm incubator at 37 ℃,5%CO2, change 2/3 solution every day.
Isolation and passage of vascular endothelial cells Under aseptic conditions，take aorta of 1-month-old rabbit (length 20 cm)， squeeze out hematocele, set in preservative fluid (0.14 M,NaCl 0.04 M,KCl,0.001 M phosphate buffer，0.001 M glucose，pH 7.4，streptomycin 100 μ/ml,penicillin 100 μ/ml)at 4 ℃. Washing with Hanks solution to no blood in artery.Hemostatic forceps clamping one end of aorta， infusing 0.1% trypsin at other end， loosing hemostatic forceps after filled，washing lumen of aorta with Hanks solution 2 times.Then clamping one end of aorta with hemostatic forceps，infusing 0.25% trypsin at other end and clamped，bathing in beaker at 37 ℃, incubating 10 min，add cell enzyme solution in beaker，washing aorta with Hanks solution，washing fluid pouring in beaker，add FCS immediately，ending action of trypsin， centrifuged for 10 min，removing supernatant，supplementing DMEM solution (15% FCS，L- glutamate 2 mmol)，mixed completely， 0.5% Trypan blue staining，counting percent of viable cells，adjusting cell number at concentration of 1×105/ml in flask，change solution every 2 days，passage and culture after growth and fusion of the cells.Remove culture solution，add 0.125%trypsin，digest and isolate， removing supernatant，add DMEM solution to produce cell suspension，passage cells to new flask by ratio of 1∶2 for culturing. Conduct experiment after passage to second generation
Effects of Different Concentration of Angiostatin on Corneal Epithelial Cells
Fibroblasts and vascular endothelial cells inoculate second generation of corneal epithelial cells in 96 well plate at concentration of 1×104/ml，divided into 4 groups，A: control group 24 well，B,C and D as study group each 24 well. Add 0.1,0.3,0.5 μg/ml AS-DMEM in B,C and D respectively after cells anchoring，at 37 ℃,5%CO2 in incubator. The changes of morphology and amount of cells after 24,48 h were observed. MTT was used to determine OD，to evaluate proliferation and inhibition of the cells.
Corneal fibroblasts and vascular endothelial cells were treated with same methods.
MTT Colorimetric Assay
Principle Dehydrogenase in viable cells can reduct tetrazolium salt into insoluble blue material - formazan,deposit in cells. The phenomenon do not occur in dead cells. DMSO can dissolve blue crystal deposited in the cell.Color depth of the solution proportional to amount of formazan.Enzyme labelled instrument was used to determine OD,to count inhibitory rate.
Inhibition rate of the cells =[(control group OD -study group OD)/control group OD]×100%。
Method 4 h before colorimetry，add MTT solution(20 μl/well)；Suction of culture solution, and then add DMSO solution (150 μl/well), at room temperature, oscillate plate on oscillator for 10 min,crystal dissolved, determine each well OD (λ=570 nm)
Statistical Analysis
Statistical software SPSS10.0,One-Way ANOVA.
RESULTS
Inhibitory effects of AS on proliferation of corneal epithelial cell, fibroblasts and vascular endothelial cell (See Table 1，2，3).
Table 1 Inhibitory Effects of AS on Proliferation of Corneal Epithelial Cells
#P>0.05，△non inhibition
Table 2 Inhibitory Effects of AS on Proliferation of Corneal Fibroblasts
*P<0.05，#P>0.05
Table 3 Inhibitory Effects of AS on Proliferation of Vascular Endothelial Cells
#P>0.05，*P<0.05
Corneal epithelial cells and vascular endothelial cells were not inhibited after AS administered 24 h, however corneal filrablasts was inhibited relatively.48 h later, results was same as 24 h, but proliferation of vascular endothelial cell was not inhibited completely. Compared to control group, viable cells were decreased markedly.
DISCUSSION
Role of Angiostatin in Inhibition of Angiogenesis
AS is one of the most powerful angiogenesis inhibitor, it can inhibit tumor angiogenesis and migration[1,2], can obtained by plasminogen degradation with MMP-2,7,9[3]. Kim, et al. found that Klinglel-3 can inhibit bFGF or VEGF induced corneal neovascularization with dose-dependent mode. Kringlel-3 can decease white blood cell significantly in cornea in study group more than that in control group.Stack MS，et al. reported that AS was one of non-competed inhibitor[3], AS can inhibit stromal increased activity of plasminogen in microcirculation of the cell result in decreasing invasive activity of the cell. It is suggests that AS mediated regulation of plasminogen formation can influence cell migration and invasive capacity. Walter，et al. confirmed that AS can inhibit proliferation of vascular endothelial cell in vitro but plasminogen cannot do that[6].So it is suggest that there are specified AS receptor in vascular endothelial cell. AS is bound with plasminogen receptor on smooth muscle, excessive plasminogen and Epsilon - aminocaproic acid (EACA) can inhibit the binding.
AS bound with plasminogen receptor can block binding of plasminogen and plasminogen activator, and block activator formation later[7]. Smooth muscle cells participating vascular forming was limited as long as lack plasminogen activator around cells[8].So, vascular epithelial cells in inhibiting vascular forming is play the roles as inhibiting proliferation and migration of vascular epithelial cells and smooth muscle cells. Moser, et al. found that AS bound with subunitα/β of ATP synthetase to down-regulate proliferation and migration of endothelial cells to mediate effects of anti-angiogenesis[9].
So, AS can inhibit neovascularization mediated by angiogenesis factor bFGF and VEGF. Gabison F, et al. confirmed that plasminogen and angiostatin are produced in the cornea. They may play a role in preventing neovascularization and may contribute to the maintenance of corneal avascularity after excimer laser keratectomy[10]. Warejcka DJ, et al. suggested that conversion of plasminogen to angiostatin is cell-type dependent. Because corneal cells generate angiostatins, use of human angiostatins may be a mean of treating abnormal corneal neovascularization without the risks of side effects[11].
Experimental Results Analysis
AS 0.1, 0.3, 0.5 μg/ ml, after 24 h without significant inhibition effects on vascular endothelial cells, after 48 h inhibition effects were occurred, viable cells were decreased markedly. But compared with same group at 24 h, viable cells were increased markedly too. It is suggested that proliferation of vascular epithelial cells was not inhibited completely.With increasing of concentration of AS, inhibitory effects were increased slightly.AS is not inhibit synthesizing DNA of vascular epithelial cells, but it is compete specific targets on the cell surface with proliferation factors to inhibit proliferation of the cells. It has inhibitory effects but do not inhibit completely without growth factors in vitro. In this study, AS 0.1,0.3 and 0.5 μg/ml cannot inhibit proliferation of corneal epithelial cells significantly.Inhibitory effects after 24 h on fibroblasts may be caused by measurement error, it is unclear on the mechanism of that effects because we are not find any inhibitory effects after 48 h.
CONCLUSION
In vitro study showed that AS 0.1,0.3,0.5μg/ml can inhibit proliferation of vascular endothelial cells slightly in the absence of growth factor. Inhibitory effects increased with increasing of concentration of AS, but never inhibit completely. And non inhibitory effects were found in corneal epithelial cells and fibroblasts. We concluded that AS may compete specific targets on the cell surface with proliferation factors to inhibit proliferation of the cells.
REFERENCES
1. O'Reilly MS, Holmgren L, Shing Y, et al. Angiostatin:a circulating endothelial cell inhibitor that suppress angiogenesis and tumor growth. Cold-Spring-Harb-Symp-Quant-Boil, 1994, 59: 471-482.
2. O'Reilly MS, Holmgren L, Chen C, et al. Angiostatin induces and sustains dormancy of human primary tumors in mice. Nat Medicine, 1996, 2: 689-692.
3. O'Reilly MS, Wiederschain D, Stetler-Stevenson WG, et al. Regulatior of angiostatin production by matrix metalloproteinase-2 in a model of concomitant resistance. J Bio Chem, 1999,274: 19568-29571.
4. Kim JH, Kim JC. Shin SH, et al. The inhibitory effects of recombinant plasogen kringlel-3 on the neovascularization of rabbit cornea induced by angiogenin, bFGF and VEGF. Exp Mol Med, 1999, 31(4): 203-209.
5. Stack MS, Gately S, Bafetti LM, et al. Angiostatin inhibits endothelial andmelanoma celhlar invasion by bloking matrix-enhanced plasogen activation. Biochem J, 1999, 340(ptl): 77-84.
6. Walter JJ, Sane DC. Angiostatin binds to smooth muscle cells in the coronary artery and inhibits smooth musc〗e cell proliferation and migration in vitro. Arterioscler Thromb Vase Biol, 1999, 19: 2041-2048.
7. Hajjar KA, Harpel PC, Jaffe EA, et al. Bindingjof plasogen to cultured human endothelial cells. JBiol Chem, 1986, 261: 11656-11662.
8. Jackson CL, Raines EW, Ross R, et al. Role of endogenous platelet-derived growth factor in arterial smooth muscle cell migration after balloon catheter injury. Arterioscler Thromb, 1993, 13: 1218-1226.
9. Moser TL, Stack MS, Asplin I, et al. Angiostatin binds ATP synthase on the surface of human endothelial cells. Proc Natl Acad Sci USA, 1999, 96(6): 2811-2816.
10. Gabison F, Chang JH, Hernandez-Quintela E, et al. Anti-angiogenic role of angiostatin during corneal wound healing. Exp Eye Res, 2004,78(3):579-589.
11. Warejcka DJ, Vauhan KA, Bernstein AM, et al. Differential conversion of plasminogen to angiostatin by human corneal cell population. Mol VIS, 2005,11:859-868.
(Editor Jaque)(LIU Hai-jun1, ZHONG Shi-z)