Role of thrombospondin-1 active fragment VR-10 synthetic peptide on rhesus choroidal-retinal endothelial cell_Chinese Journal of Ocular Fundus Diseases

Authors：

TianRun ,  MeiYan , ChenZhen , 方林 , 李小芹

Department of Ophthalmology, The First People's Hospital of Yunnan Province, Kunming 650032, China;

Corresponding author：

MeiYan, Email: meikm@163.com

Keywords：

Thrombospondin 1/drug effects; Endothelial cells/pathology; Cell proliferation; Cell migration inhibition

DOI：

10.3760/cma.j.issn.1005-1015.2015.03.015

Video：

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Objective To investigate the effects of thrombospondin-1 active fragment (TSP-1) synthetical peptide VR-10 on proliferation and migration of rhesus choroidal-retinal endothelial (RF/6A) cell and the expressions of apoptosis relative genes in RF/6A cell. Methods The survival rate of RF/6A cell were detected by methyl thiazolyl tetrazolium, and migration ability was measured by transwell chamber after exposure to 1.0 μg/ml TSP-1 and synthetic peptide VR-10 (0.1, 1.0, 10.0 μg/ml) for different times (6, 12, 24, 48 hours). Caspase-3 and factor associated suicide (FAS) protein levels were measured by Western blot. The mRNA level of bcl-2 and FAS ligand (FASL) were measured by reverse transcription-polymerase chain reaction (RT-PCR). Results The survival rate of RF/6A cells was determined by the treatment time and concentration of TSP-1(1.0 μg/ml) and the synthetic peptide VR-10 (0.1, 1.0, 10.0 μg/ml). The lowest survival ratio of RF/6A was 78% (P < 0.001) when cells were treated by 10 μg/ml synthetic peptide VR-10 after 48 hours. TSP-1 and synthetic peptide VR-10 could inhibit migration of RF/6A cells in transwell chamber (P < 0.001). 10.0 μg/ml synthetic peptide VR-10 had the strongest effect, 1.0 μg/ml TSP-1 was the next. Migration inhibition rate was increase with the increase of the concentration of VR-10 (P < 0.001). There was no significant differences between 0.1 μg/ml and 1.0 μg/ml VR-10 (P=0.114). Western bolt showed that RF/6A cell in control group mainly expressed the 32×10³ procaspase-3 forms. To 10.0 μg/ml VR-10 treated group, it showed decreased expression of procaspase-3 (32×10³) and concomitant increased expression of its shorter proapoptotic forms (20×10³). Compared with control group, expression of FAS peptides were significantly increased in 10.0 μg/ml VR-10 treated group. Compared with control group, expression of FasL mRNA was significantly increased in 10.0 μg/ml VR-10 treated group(t=39.365, P=0.001), but the expression of bcl-2 mRNA was decreased(t=-67.419, P=0.000). Conclusion TSP-1 and synthetic peptide VR-10 had the ability to inhibit proliferation and migration of endothelial cell, and also induce apoptosis by increasing FAS/FASL expression and repressing bcl-2 expression.

Citation： TianRun, MeiYan, ChenZhen. Role of thrombospondin-1 active fragment VR-10 synthetic peptide on rhesus choroidal-retinal endothelial cell. Chinese Journal of Ocular Fundus Diseases, 2015, 31(3): 274-278. doi: 10.3760/cma.j.issn.1005-1015.2015.03.015 Copy

1.	田润, 唐罗生, 梅妍, 等.血小板反应蛋白-1在缺氧人视网膜色素上皮细胞中的表达[J].中华眼底病杂志, 2011, 27(4):385-386.
2.	Sorenson CM, Wang S, Gendron R, et al.Thrombospondin-1 deficiency exacerbates the pathogenesis of diabetic retinopathy[J/OL]. J Diabetes Metab, 2013, 12(Suppl):1[2013-05-25].http://europepmc.org/abstract/MED/24224119.
3.	Soriano-Romaní L, García-Posadas L, López-García A, et al.Thrombospondin-1 induces differential response in human corneal and conjunctival epithelial cells lines under in vitro inflammatory and apoptotic conditions[J].Exp Eye Res, 2015, 134:1-14.
4.	Fei P, Zaitoun I, Farnoodian M, et al.Expression of thrombospondin-1 modulates the angioinflammatory phenotype of choroidal endothelial cells[J/OL].PLoS One, 2014, 9(12):116423[2014-12-30]. http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0116423.
5.	Farnoodian M, Kinter JB, Yadranji Aghdam S, et al.Expression of pigment epithelium-derived factor and thrombospondin-1 regulate proliferation and migration of retinal pigment epithelial cells[J/OL].Physiol Rep, 2015, 3(1):12266[2015-01-01]. http://physreports.physiology.org/content/3/1/e12266.long.
6.	Adams JC, Tucker RP. The thrombospondin type 1 repeat (TSR) superfamily:diverse proteins with related roles in neuronal development[J]. Dev Dyn, 2000, 218(2):280-299.
7.	宋忻, 韩忠朝.凝血酶敏感蛋白-1(TSP-1)的结构与功能研究进展[J].医学分子生物学杂志, 2005, 2(1):45-48.
8.	Lawler J, Simons E. Cooperative binding of calcium to thrombospondin[J]. J Biol Chem, 1983, 258(20):12098-12101.
9.	Volpert OV, Zaichuk T, Zhou W, et al. Inducer-stimulated Fas targets activated endothelium for destruction by anti-angiogenic thrombospondin-1 and pigment epithelium-derived factor[J]. Nat Med, 2002, 8(4):349-357.
10.	Dawson DW, Volpert OV, Pearce SFA, et al. Three distinct D-amino acid substitutions confer potent antiangiogenic activity on an inactive peptide derived from a thrombospondin-1 type 1 repeat[J]. Mol Pharmacol, 1999, 55(2):332-338.
11.	Bagavandoss P, Wilks JW. Specific inhibition of endothelial cell proliferation by thrombospondin[J]. Biochem Biophys Res Commun, 1990, 170(2):867-872.
12.	Yafai Y, Eichler W, Iandiev I, et al. Thrombospondin-1 is produced by retinal glial cells and inhibits the growth of vascular endothelial cells[J].Ophthalmic Res, 2014, 52(2):81-88.
13.	付永锋, 樊廷俊. Bcl-2家族蛋白与细胞凋亡[J].生物化学与生物物理学报, 2002, 34(4):389-394.
14.	Nör JE, Mitra RS, Sutorik MM, et al. Thrombospondin-1 induces endothelial cell apoptosis and inhibits angiogenesis by activacting the caspase death pathway[J]. J Vasc Res, 2000, 37(3):209-218.
15.	Shimonishi T, Isse K, Shibata F, et al.Up-regulation of fas ligand at early stages and down-regulation of Fas at progressed stages of intrahepatic cholangiocarcinoma reflect evasion from immune surveillance[J].Hepatology, 2000, 32(4 Pt 1):761-769.

1. 田润, 唐罗生, 梅妍, 等.血小板反应蛋白-1在缺氧人视网膜色素上皮细胞中的表达[J].中华眼底病杂志, 2011, 27(4):385-386.
2. Sorenson CM, Wang S, Gendron R, et al.Thrombospondin-1 deficiency exacerbates the pathogenesis of diabetic retinopathy[J/OL]. J Diabetes Metab, 2013, 12(Suppl):1[2013-05-25].http://europepmc.org/abstract/MED/24224119.
3. Soriano-Romaní L, García-Posadas L, López-García A, et al.Thrombospondin-1 induces differential response in human corneal and conjunctival epithelial cells lines under in vitro inflammatory and apoptotic conditions[J].Exp Eye Res, 2015, 134:1-14.
4. Fei P, Zaitoun I, Farnoodian M, et al.Expression of thrombospondin-1 modulates the angioinflammatory phenotype of choroidal endothelial cells[J/OL].PLoS One, 2014, 9(12):116423[2014-12-30]. http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0116423.
5. Farnoodian M, Kinter JB, Yadranji Aghdam S, et al.Expression of pigment epithelium-derived factor and thrombospondin-1 regulate proliferation and migration of retinal pigment epithelial cells[J/OL].Physiol Rep, 2015, 3(1):12266[2015-01-01]. http://physreports.physiology.org/content/3/1/e12266.long.
6. Adams JC, Tucker RP. The thrombospondin type 1 repeat (TSR) superfamily:diverse proteins with related roles in neuronal development[J]. Dev Dyn, 2000, 218(2):280-299.
7. 宋忻, 韩忠朝.凝血酶敏感蛋白-1(TSP-1)的结构与功能研究进展[J].医学分子生物学杂志, 2005, 2(1):45-48.
8. Lawler J, Simons E. Cooperative binding of calcium to thrombospondin[J]. J Biol Chem, 1983, 258(20):12098-12101.
9. Volpert OV, Zaichuk T, Zhou W, et al. Inducer-stimulated Fas targets activated endothelium for destruction by anti-angiogenic thrombospondin-1 and pigment epithelium-derived factor[J]. Nat Med, 2002, 8(4):349-357.
10. Dawson DW, Volpert OV, Pearce SFA, et al. Three distinct D-amino acid substitutions confer potent antiangiogenic activity on an inactive peptide derived from a thrombospondin-1 type 1 repeat[J]. Mol Pharmacol, 1999, 55(2):332-338.
11. Bagavandoss P, Wilks JW. Specific inhibition of endothelial cell proliferation by thrombospondin[J]. Biochem Biophys Res Commun, 1990, 170(2):867-872.
12. Yafai Y, Eichler W, Iandiev I, et al. Thrombospondin-1 is produced by retinal glial cells and inhibits the growth of vascular endothelial cells[J].Ophthalmic Res, 2014, 52(2):81-88.
13. 付永锋, 樊廷俊. Bcl-2家族蛋白与细胞凋亡[J].生物化学与生物物理学报, 2002, 34(4):389-394.
14. Nör JE, Mitra RS, Sutorik MM, et al. Thrombospondin-1 induces endothelial cell apoptosis and inhibits angiogenesis by activacting the caspase death pathway[J]. J Vasc Res, 2000, 37(3):209-218.
15. Shimonishi T, Isse K, Shibata F, et al.Up-regulation of fas ligand at early stages and down-regulation of Fas at progressed stages of intrahepatic cholangiocarcinoma reflect evasion from immune surveillance[J].Hepatology, 2000, 32(4 Pt 1):761-769.

Chinese Journal of Ocular Fundus Diseases

Role of thrombospondin-1 active fragment VR-10 synthetic peptide on rhesus choroidal-retinal endothelial cell

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