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Subrata Das, Derick G Holt, Hironori Uehara, Xiaohui Zhang, Bonnie Archer, Balamurali K. Ambati; Raver2 preserves corneal avascularity by increasing sFlt1 Production. Invest. Ophthalmol. Vis. Sci. 2014;55(13):3249.
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© ARVO (1962-2015); The Authors (2016-present)
Neovascularization (NV) is a serious complication of many leading causes of vision loss. sFlt1 is an important endogenous regulator of angiogenesis that arises from alternative splicing of FLT1. The sFlt1 acts as a “decoy” that prevents VEGF angiogenic signaling. Our previous work showed that sFlt1 is the key preserver of corneal avascularity and loss of sFlt1 is associated with pathologic corneal neovascularization (KNV). However, mechanisms underlying the production of sFlt1 have not been identified. Our studies investigate Raver2, a nuclear protein play an impotent role in the molecular mechanisms of the Flt1 RNA splicing event.
Raver2 knock-down was done with corneal plasmid (Raver2 shRNA ) injectionand Raver2-specific siRNAs transfection in HUVEC and HeLa cloned with mouse Flt minigene. For over expression, FLAG-tagged raver2 plasmid was also transfected in HUVEC and injected in Pax6+/- mice cornea. mRNA levels were assayed by RT-PCR and protein levels determined by Western and ELISA. Rescue experiment was performed by Raver2 siRNA following Raver2 recombinant protein transfection in mouse Flt mini gene cloned HeLa. Molecular associations were investigated by IP (Imunoprecipitation), Co-IP and RNA Immunoprecipitation (RIP).
Knock-down of Raver2 decreased sFlt1 levels significantly both in vivo (mouse cornea) and in vitro (HUVEC and Flt mini gene cloned HeLa). We also observed marked KNV following Raver2 knock-down in WT mice. While Raver2 overexpression suppressed spontaneous, KNV in Pax6+/- mice. Given the functional importance of Raver2, we sought to determine if the factor acted at the level of Flt1 pre-mRNA. RIP experiments showed enrichment of Flt1 RNA, implicating a direct association of Raver2 with Flt1 pre-mRNA. To further characterize Raver2 function, IP with Raver2 showed specific enrichment for PTB, a well-studied factor involved in alternative splicing. RIP with PTB also enriched Flt1 RNA and RIP with PTB following Raver2 knock-down showed significantly reduced PTB occupancy at FLT1 pre-mRNA.
Our data suggested that Raver2 promotes sFlt1 production by interacting with polypyrimidine tract-binding protein (PTB) and a segment of Flt-1 pre-mRNA. This model will provide an opportunity to manipulate alternative splicing of FLT1 to promote an anti-angiogenic therapy.
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