The Effects Of CD105 Gene Silencing To Experimental Choroidal Neovascularization In BN Rats

BackgroundsChoroidal neovascularization(CNV) is the main cause of severe vision loss associated age-related macular degeneration(AMD) and some other fundus diseases.Choroidal neovascularization(CNV) also named subretinal neovascularization.It derived from choroidal vessels and breaks through Bruch's membrane then grows into the subretinal pigmental epithelial space.Because of higher permeability of the new vessels wall,it cause exudation,hemorrhage,cicatrix forming and impair central vision at last. CNV is the leading cause of blindness in individuals aged 55 years and older in developed countries.How to inhibit CNV is a problem to ophthalmologists and researchers for a long time.The exact mechanism of CNV is not clear,there is no ideal therapy to it.The outcomes of current treatments such as Laser photocoagulation, PDT(photodynamic therapy),TTT(through pupil thermotherapy), surgery excision,radiotherapy are not satisfactory for recurrence or additional damage to ocular tissues.Gene therapy provides prospect to treatment of CNV.Anti-VEGF (vascular endothelial growth factor) agents have dramatically changed the treatment and prognosis of patients with CNV.VEGF and it's receptor expressed widely at ocular tissues and it is proved that normal level of VEGF expression is vital for photoreceptor survive and maintaining chorodial vasculature.Long term use of anti-VEGF agents may has potential risk.Seek new target and method is necessary.CD105 is a TGF-βsuperfamily receptor and expressed predominantly on vascular endothelial cells.CD 105 was regard as a specific marker of proliferating endothelium.Researches showed CD105 is essential for endothelium angiogenic activity and it may plays a critical role in CNV.CD105 can be a target for anti- angiogenesis therapy.Objective1.Estabish the CNV model in BN(brown norway) rats,then investigate the expression of CD105 and VEGF(vascular endothelial growth factor) in it.2.Construct the recombinant plasmids expressing CD105 short hairpin RNA(shRNA) by Pgenesil-1 plasmid,and to screen the highly efficient shRNA in BN rat CNV model.3.Research the effect of CD 105 gene silencing to CNV on BN rat CNV model.Methods1.Choroidal neovascularization was induced in Brown Norway rats by diode-laser photocoagulation.The formation and nature process of CNV was evaluated by fundus fluorescein angiography on 0,3,7,14, 21and 28 days after photocoagulation.Light microscopy examination and quantitive analysis of CNV was performed on 14 days.2.The expression of CD105 was evaluated by immunohistochemistry and semi-quantitive reverse transcription polymerise chain reaction in CNV model,and the relationship between CD105 and CNV was studied. Four recombinant Pgenesil-1 with U6 promoter was constructed based on the segnence information of CD105,three targeting rat CD105 and one unspecific.Metafectene containing Pgenesil-1 was injected into subretinal space in the eyes of BN rat.Localization of GFP was oberserved by fluorescence microscopy.Three recombinant plasmids being transfected into BN rat CNV model,the expression of CD105 mRNA level was determined by reverse transcription polymerise chain reaction 2 weeks later.3.The effective CD105 shRNA express plasmid was transinfectd in BN rat CNV model by subretina injection.The result of CD105 gene silencing to CNV model was evaluated dy FFA,quantitive analysis of CNV and reverse transcription-polymerise chain reaction.The expression of VEGF at mRNA level was evaluated by reverse transcription-olymerise chain reaction.Result1.Diode laser photocoagulation on BN rat's retina can induce the CNV successfully.The CNV occurred on day 7,reached the peak on day 14 and sustained more than 28 day after photocoagulation.CNV was confirmed by light microscopy examination and quantitive analysis.2.CD105 expresses weakly at mRNA and protein level in normal BN rats retina.In CNV model,CD105 expression increased at 7day, gradually increased to 21 day,and decreased at 28 day after photocoagulation.3.One day after transfection,green fluorescence was observed in the rat's retina including RPE cells under fluorescent microscope.The intensity became stronger on the second and third week than the first week,and sustained four weeks.The CD105 shRNAs were successfully inserted into plasmid Pgenesil-1.The recombinants were identified by sequencing.Compared with the controls,the expression of CD105 mRNA level in BN rat CNV model transfected with CD105 shRNA recombinants was markedly down-regulated 78±5%(P＜0.01) and 52±3%(P＜0.01) respectively in two groups of recombinant plasmids, whereas it had not any significant changes in another shRNA and unspecific shRNA-transfected and only with transfection reagent to BN rat CNV model.4.In CD105shRNA-treated group and CNV model control group, the incidence of fluorescein leakage was 24.6%and 63.2%at 14 day after photocoagulation.Significant difference was found between the two groups.The result of quantitive analysis of CNV showed the area of CNV leakage is(14.46±0.82)×10~3μm~2and(31.22±1.46)×10~3μm~2 in CD105 shRNA-treated group and CNV model control group,significant difference was found between the two groups.The expression of VEGF at mRNA level in CNV model control group reached the peak at 7 day after photocoagulation,then decreased gradually,at 28 day it decreased significantly.It is similar in CD105shRNA-treated group,but the expression decreased significantly at each time point.At 14 day,the expression of VEGF at mRNA level in CD105shRNA-treated group is 36.7%of it in CNV model control group.The expression of CD105 at mRNA level in CNV model control group was found at 7 day after photocoagulation,reached the peak at 14 day,and then decreased gradually,at 28 day it decreased significantly.It is similar in CD105 shRNA-treated group,but the expression decreased significantly at each time point.At 14 day,the expression of VEGF at mRNA level in CD105shRNA-treated group is 21.68%of it in CNV model control group.Conclusion1.To estabish the CNV animal model by diode laser photocoagulation on BN rat's retina is feasible.2.The expression pattern of CD105 in CNV is consistent to the formation of CNV.CD105 is an important marker of CNV.3.Metafectene can transfect Pgenesil-1 into the retina of the BN rat effectively,and their expression can maintain 4 weeks after transfection.4.Three recombinants expressing CD105 shRNA are successfully constructed.Pgenesil-ENG2 could silence CD105 expression in vivo potently.5.CD105 gene silencing at early stage could inhibit the formation of CNV in vivo.Down-regulation of VEGF expression may be one of it's mechanisms.6.CD105 plays an important role in early stage of CNV,it is hopeful to be a target of CNV therapy.