Fbln5-RPE Inhibits CNV Of AMD And Its Mechanism

IntroductionAge-related macular degeneration (AMD) is the most common cause of irreversiblevision loss, affecting more than1.75million people in the United States and25to30million worldwide. Choroidal neovascularization (CNV), the abnormal angiogenesis of thechoroidal vasculature via Bruch's membrane, is the predominant cause of severe visual lossin AMD. CNV is a multistep process, beginning with the breakdown of Bruch's membrane,followed by choroidal endothelial cells (CECs) migration from the choroid into the retinalpigment epithelium (RPE) and/or sub-retinal space, and finally CECs proliferation andtubulogenesis. Thus, CECs migration and proliferation play a critical role in thedevelopment of CNV in AMD. However, the mechanisms responsible for the control ofCECs migration and proliferation are not completely understood.Vascular endothelial growth factor (VEGF) has been identified as a key angiogenicfactor responsible for CNV. Multiple mechanisms have been proposed to explicate the roleof VEGF in the pathogenic progression of CNV, a process that includes CECs proliferation,survival, and migration, the release of matrix metalloproteinases and increased permeability,and integrin turnover with endothelial migration. Several anti-VEGF therapeutic strategieshave been developed and have proved effective for controlling neovascular AMD, butdespite the clinical benefits these strategies are also challenged by patient'sunresponsiveness, drug resistance, need for repetitive delivery, and adverse side effects.Therefore, a need still exists for new targeted therapies for AMD.Fibulin5(FBLN5) is an extracellular matrix glycoprotein that is essential forelastogenesis. Recent findings have implicated Fibulin5in the pathogenesis of AMD.FBLN5is expressed in the retina, RPE, and Bruch's membrane, the intercapillary pillars ofthe choriocapillaris in normal eyes and in the pathologic basal deposits beneath the RPE ineyes with AMD. Missense mutations in the Fbln5gene have been associated with increasedsusceptibility to AMD. In mice and humans without functional copies of the Fbln5gene,the assembly of elastin within Bruch's membrane is altered. These findings suggest that FBLN5may be involved in maintaining the integrity of Bruch's membrane through theassembly and stabilization of extracellular matrix structures.Additionally, FBLN5also functions as an endogenous angiogenesis inhibitor and isimplicated in regulating CNV. It not only acts as a bridging peptide between elastin fibersand cell surface integrins in the blood vessel wall but also interacts with VEGF andinterferes with CNV. Furthermore, it was found that some Fbln5missense mutationsappeared to alter VEGF regulation, reducing FBLN5secretion and leading to unregulatedblood vessel growth. Thus, FBLN5plays a pleiotropic role in the pathogenesis of AMD.Since CNV, structural alterations in Bruch's membrane and retinal pigment epithelial(RPE) dysfunction play a critical role in the pathogenesis of neovascular AMD, the specificinhibition of these processes should prevent and reverse the progression of thisvision-threatening disease. Given the multifunctional nature of FBLN5and RPE in thepathogenesis of AMD, it is a promising target for therapeutic intervention. So, we will usethe Overexpression of Fibulin5in RPE transplantation to inhibit CNV of AMD.Objective: Age-related macular degeneration (AMD) is the most common cause ofirreversible vision loss. FBLN5plays a pleiotropic role in the pathogenesis of AMD. Weexamined whether the in vitro overexpression of FBLN5in retinal pigment epithelial (RPE)cells alters the proliferation and migration of cocultured choroidal endothelial cells (CECs)and explored the possible mechanisms involved. Then, transplanted overexpression ofFBLN5in RPE into subretinal space of laser-induced CNV model to evaluate CNVchanges in vivo.Materials and Methods:1. A recombinant lentiviral vector carrying the Fbln5gene of Fbln5full-length cDNAplasmid (pExpress-1-Fbln5) was generated to transduce rat RPE cells, which were dividedinto GFP unloaded transfected RPE (GFP-RPE) group (negative control) and Fbln5genetransduced RPE (Fbln5-RPE) group (experimental group). The expression of FBLN5intransduced RPE cells was detected by quantitative real-time PCR and Western blot.2. The transduced RPE cells were then cocultured with rhesus macaque CECs in aTranswell coculture system. RPE only, GFP-RPE and Fbln5-RPE were used to coculturedwith CECs.The impact of overexpression of FBLN5in RPE cells on CECs proliferation andmigration was assessed, as well as the impact on the mRNA expressions of vascular endothelial growth factor (VEGF), C-X-C chemokine receptor type4(CXCR4), andtransforming growth factor β1(TGFB1).3. Laser induced LE rat CNV model was established. Then transduced RPE wastransplanted into subretinal space of this CNV model, and leakage area of fluorescein inFundus Fluorescein Agiography(FFA) and CNV counting were measured. Assay wasdivided into GFP-RPE group (negative control) and Fbln5-RPE group (experimental group).FFA was performed on1,2,3and4weeks post-operation and pre-operation respectively.Results:Part Ⅰ1. Our results showed that a recombinant lentivirus carrying the Fbln5gene, whichcould induce overexpression of FBLN5in RPE cells, was successfully conctructed. Theresults of vectors sequencing were completely correct. The efficiency of infection in RPE isabout70-80%.2. Primary culture of LE rats RPE was stabilized.90%cells were RPE cells whichwere verified by its pigmentation and hexagonal morphology characteristcs, and byspectrum keratin DAB identification. As passages went on, their pigments became less andmorphology showed spindle-shaped cells.3. Overexpression of Fbln5in RPE was detected by Q-PCR and WB, and showed aefficient overexpression and secretion out of cells which were256times compared to thecontrol group.Part Ⅱ1. Migration inhibition test found that overexpression of FBLN5can significantlyinhibit the CECs migrate through the Transwell chamber by cell counting and ODmeasurement (n=5, P <0.01). There was no significant difference between the negativecontrol group and blank control group.2. On1,2,3days after coculture, MTT results showed that overexpression of FBLN5could significantly inhibit OD values of CECs.(n=5, P <0.05).No statistical differencebetween the negative control group and blank control group (n=5, P>0.05).3. On1,2,3days after coculture, cell cycle measurement showed that overexpressionof FBLN5can significantly inhibit the proportion of the CECs cells in S phase andproliferation index has been significantly inhibited (n=3, P <0.05). No statistical difference between the negative control group and blank control group (n=3, P>0.05). Each timepoint CECs apoptosis coefficient between the three groups were not statistically different (n=3, P>0.05).4. On1,2,3days after coculture, the Q-PCR showed that over-expression of FBLN5downregulated the mRNA expressions of VEGF, CXCR4, and TGFB1in cocultured CECs(n=3,P <0.01).Part Ⅲ1. A laser-induced LE CNV model in rats was built, and FFA was performed on1-5weeks after laser. FFA showed that about fluorescein leakage presented in70%of the laserspot at the same time frozen sections of retina showed CNV formation.2. On1,2,3and4weeks after transplantation, frozen sections of retina showed retinaledema and GFP green fluorescence positive cells in subretinal space. On2,3, and4weeksafter transplantation, the CNV leakage area ratio and CNV counting ratio showedsignificant difference between two groups (n=3, P <0.05). Overexpression of FBLN5inRPE inhibited CNV significantly in vivo.Conclusions:1. A pZlen-Fbln5-IRES-GFP-RPE letivirus overexpression system was conctructedsuccessfully, and FBLN5was overexpressed and secreted extracellular efficiently.2. In coculture of CECs and RPE cells, FBLN5overexpressed RPE cells inhibited cellproliferation and migration of CECs, the mRNA level of VEGF and CXCR4weredownregulated, which maybe the pathway of the inhibitory effect of Fbln5-RPE on CECs.3. Transplantation of Fbln5-RPE in the retina of laser-induced CNV LE rats couldinhibit leakage area and the numbers of CNV. It implis that FBLN5may work as a potentialtarget for therapeutic intervention in neovascular in AMD.