Cloning,Expression And Functional Study Of Novel Platelet Derived Growth Factor C Splicing Variants Isolated From Fibrovascular Membranes Of Proliferative Diabetic Retinopathy Patients

Background:The anti-Vascular Endothelial Growth Factor(VEGF)therapy has gradually become the first-line medicine for treatment of proliferative diabetic retinopathy(PDR),however,a considerable number of patients do not respond to anti-VEGF therapy.Based on the literature report,Platelet Derived Growth Factor C(PDGFC)induces retinal and choroidal neovascularization via a VEGF-independent pathway in rodent models,hence attracts significant attention as an important gene target other than VEGF.Notwithstanding the expression and regulatory mechanisms of PDGFC in fibrovascular membranes of proliferative diabetic retinopathy(PDR)patients has never been studied.Purpose:To detect the expression of PDGFC in fibrovascular membranes of PDR patients,isolate and clone novel PDGFC splicing variants from the membranes as well as study their expression and function in retinal microvessel endothelial cells.Methods:Fibrovascular membranes were collected during vitrectomy from eight patients diagnosed with PDR.The total RNA was extracted from the membranes and reverse transcribed into cDNA.Multiple PDGFC fragments were amplified by regular PCR using the cDNA as the template and the primers corresponding to the full-length PDGFC.The PCR products were cloned into TOPO-TAPCR2.1 vector for sequencing.Sequence blast search was performed to confirm the identities of the positive clones as two novel splcing variants and full length of PDGFC,respectively.The cDNA encoding both full-length and splicing variants of PDGFC were subcloned into pcDNA3.0 expression vector and designated as p-PDGFCfull,p-PDGFC1,and p-PDGFC2,respectively.FLAG tag was also added to the C-terminus of these protein-encoding sequences to facilitate detection.The endotoxin-free plasmid of p-PDGFCfull,p-PDGFC1,p-PDGFC2,and pcDNA vecror were either transfected alone or co-transfected with Green Florescent Protein(GFP)-expressing plasmid into monkey RF/6A retinal microvessel endothelial cells.The transfection efficiency was estimated by co-transfection with GFP-expressing plasmid or examined by genomic real-time PCR.The expression and cellular location of PDGFCfull,PDGFC1,and PDGFC2 proteins were detected by immunoflorescence.The functional study was performed by examining the proliferation of the retinal microvessel endothelial cells using Cell Counting Kit detecting the total PDGFC transcript levels(exogenous and endogenous)by real-time PCR.Results:PCR products with variable length were observed in agarose gel electrophoresis.Sequencing results demonstrate homology of the positive clones to human PDGFC.Further analyses revealed that these products contain both full-length PDGFC and two novel splicing variants that result in two truncated proteins predicted by open reading frame analysis.Both full-length and splicing variant protein encoding sequences were then cloned into pcDNA3.0 expression vector,and restriction enzyme digestions were performed to confirm the positive clones.The plasmid p-PDGFCfull,p-PDGFC1 and p-PDGFC2 were then transfected into the retinal microvessel endothelial cells,the full-length and truncated PDGFC proteins were predominantly localized to the cytoplasm of the endothelial cells.Functionally speaking.with the comparable transfection efficiency,the endothelial cells transfected with p-PDGFC1 and p-PDGFC2 exhibited significantly reduced proliferation(p<0.001 for p-PDGFCl transfected cells,p<0.05 for p-PDGFC2 transfected cells),whereas those transfected with p-PDGFCfull showed a significantly increased proliferation(p<0.001),as compared to the cells transfected with pcDNA empty vector,respectively,and the cell proliferation induced by p-PDGFCfull transfection could be abrogated by co-transfection of p-PDGFC1 and p-PDGFC2(both p>0.05).Furthermore,the results of real-time PCR revealed a similar trend of total PDGFC mRNA levels among the transfection groups.Conclusion:Two novel splicing variants of PDGFC were identified in fibrovascular membranes of PDR patients,and were then cloned into pcDNA vector and expressed in non-human primate retinal microvessel endothelial cells.The expression of the PDGFC splicing variants resulted in a significant reduction in cell number and aboragated the cell proliferation induced by over-expression of full-length PDGFC.These results suggest a negative feedback regulatory mechanism underlying PDGFC expression that might exist in fibrovascular membranes of PDR patients,in other words,under the pathological condition of PDR,the full length PDGFC was generated in the presence of two novel splicing variants,both of which could function to antagonize the full-length PDGFC induced neovascularization,possibly via the machnism of down-regulating total PDGFC expression.More importantly,the identification,cloning and expression of the novel splicing variants provide theoretical and experimental basis on which new therapeutic intervention for neovascularization in PDR will developed.