BRIEF DESCRIPTION OF DRAWINGS
FIG. 1, panel A is a photo of a human eye demonstrating abrupt termination of blood vessels in the conjunctiva (CJ) at its border with the cornea (C), the limbus
FIG. 1, panel B depicts a non-reducing western blot of mouse cornea revealing immunoreactive bands of VEGF-A at 100-130 kDa corresponding to bound forms and negligible immunoreactivity at 45-50 kDa corresponding to the free form.
FIG. 1, panel C depicts sflt-1 (lane 1) and VEGF-A (lane 3) transcripts in mouse cornea identified by representative RT-PCR. Lane 2 is water (template negative) control.
FIG. 1, panel D and panel E depict sflt-1 mRNA detected by in situ hybridization in mouse corneal epithelium (epi) and stroma (str). Antisense RNA probes show purple-brown reactivity. Sense RNA probes show negligible reactivity.
FIG. 1, panel F depicts immunolocalization (brown) of sflt-1 protein in mouse cornea.
FIG. 1, panel G depicts reducing western blots using an antibody against the amino (N)-terminus of flt-1 that recognizes both mbflt-1 and sflt-1 and an antibody against the unique carboxyl terminus of sflt-1 specific antibody reveal that mouse cornea (1) contains primarily sflt-1 (60 kDa) while conjunctiva (2) contains primarily mbflt-1 (190 kDa).
FIG. 1, panel H depicts western blot analysis of two independent mouse cornea samples immunoprecipitated with anti-VEGF-A antibody and immunoblotted with a biotinylated antibody against the amino (N)-terminus of flt-1 that recognizes both mbflt-1 and sflt-1 reveals that VEGF-A interacts with sflt-1 (60 kDa). Subsequent immunoblot with a biotinylated anti-VEGF-A antibody confirms the pull-down of VEGF-A by the immunoprecipitating antibody.
FIG. 2, panel A depicts topical enzymatically active Cre recombinase abolishes corneal avascularity in flt-1loxP/loxP mice. NLS-Cre induces Cre expression (brown) in the cornea within 1 hour of eye drop application as demonstrated by immunolocalization in cell nuclei.
FIG. 2, panel B shows that NLS-β-galactosidase expression fails to induce Cre expression (brown) in cornea within 1 hour of eye drop application as demonstrated by immunolocalization in cell nuclei stained red.
FIG. 2, panel C depicts a reducing western blot of Cre expression.
FIG. 2, panel D depicts Xgal staining of corneal flat mount of ROSA26R lacZ reporter mouse confirms expression of β-galactosidase (blue) 2 days after Cre expression.
FIG. 2, panel E depicts representative corneal flat mounts showing CD31+ (green) LYVE-1− blood vessels in flt-1loxP/loxP mouse corneas 14 days after treatment with NLS-Cre eye drops.
FIG. 2, panel F depicts representative corneal flat mounts showing CD31+ (green) LYVE-1 blood vessels in flt-1loxP/loxP mouse corneas 14 days after treatment with NLS-β-galactosidase expression.
FIG. 2, panel G depicts the absence of corneal vascularization in wild-type mice after topical application of NLS-Cre.
FIG. 2, panel H depicts the absence of corneal vascularization in wild-type mice after topical application of NLS-β-galactosidase.
FIG. 2, panel I depicts a western blot indicating that topical application of NLS-Cre leads to Cre expression in the mouse cornea and is enhanced by the prior topical application of proparacaine eye drops (+P) compared to no prior application (−P).
FIG. 3, panel A is a bar graph indicating that sflt-1 mRNA knockdown abolishes corneal avascularity. Real time RT-PCR reveals reduced sflt-1 mRNA in wild-type mouse corneas 3 days after injection of pshRNA-sflt-1 but not pshRNA-mbflt-1. * P<0.05, Bonferroni corrected Mann Whitney U test. Error bars depict s.e.m.
FIG. 3, panel B is a bar graph depicting data generated from an ELISA. The data indicates that sflt-1 protein is reduced in wild-type mouse corneas 3 days after injection of pshRNA-sflt-1 but not pshRNA-mbflt-1. * P<0.05, Bonferroni corrected Mann Whitney U test. Error bars depict s.e.m.
FIG. 3, panel C is a bar graph depicting data generated from an ELISA. The data indicates that free VEGF-A protein is increased in wild-type mouse corneas 3 days after injection of pshRNA-sflt-1 but not pshRNA-mbflt-1. * P<0.05, Bonferroni corrected Mann Whitney U test. Error bars depict s.e.m.
FIG. 3, panel D provides an image of an eye expressing pshRNA-sflt-1.
FIG. 3, panel E depicts a corneal flat mount showing CD31+ (green) LYVE-1− blood vessels at 14 days after injection. pshRNA-sflt-1 expression induces CV in wild-type mice. Scale bars are 500 μm.
FIG. 3, panel F depicts a corneal flat mount showing CD31+ (green) LYVE-1− blood vessels at 14 days after injection. pshRNA-mbflt-1 expression fails to induce CV in wild-type mice. Scale bars are 500 μm.
FIG. 3, panel G is a graph depicting a decrease in sflt-1 (sVEGFR1) expression by a siRNA sequence that targets sflt-1 (sVEGFR1).
FIG. 3, panel H depicts suppression of unique tail of sFLT by siRNA targeting unique tail.
FIG. 3, panel I depicts suppression of mRNA of VEGF binding domains (553 bp) of sFLT by siRNA targeting unique tail, while 18sRNA (315 bp) is unaffected.
FIG. 3, panel J depicts western blot analysis of VEGF expression (band visible at 25 kD) in mouse corneas using immunoprecipitation by antibody to sFLT unique tail and demonstrating that siRNA knock down of sflt-1 expression frees VEGF from sflt-1 sequestration.
FIG. 4, panel A provides reducing western blots showing a deficiency of sflt-1 in corneas of corn1 and Pax6+/− mice compared to background strain A/J and Pax6+/+ mice.
FIG. 4, panel B is a bar graph showing that sflt-1/Fc administration inhibits CV in corn1 and Pax6+/− mice compared to IgG1/Fc (by 87±2% in corn1; P=0.01; by 85±3% in Pax6+/−; P=0.03) and to control untreated mice (by 87±2% in corn1; P=0.01; by 84±3% in Pax6+/−; P=0.03). Significance by Bonferroni corrected Mann Whitney U test. Error bars depict s.e.m.
FIG. 4, panel C depicts flat mounts showing CD31+ (green) LYVE-1—corneal blood vessels.
FIG. 4, panel D depicts immunostaining of a cornea and revealing a deficiency of sflt-1 (brown) in cornea with aniridia-associated vascularization (top), revealed by vascular cell adhesion molecule-1 (VCAM-1) staining (red) compared to the avascular cornea (lack of VCAM-1 staining) of a different cornea without aniridia (bottom).
FIG. 4, panel E depicts a marked deficiency of sflt-1 (reddish brown) staining in cornea of Antillean manatee.
FIG. 4, panel F depicts the presence of sflt-1 (reddish brown) staining in cornea of a dugong.
FIG. 4, panel G depicts the presence of sflt-1 (reddish brown) staining in cornea of an African elephant.
FIG. 4, panel H depicts the presence of sflt-1 (reddish brown) staining in cornea of a beaked whale.
FIG. 4, panel I depicts reducing western blots using an antibody against the amino (N) terminus of flt-1 reveal presence of sflt-1 (60 kDa) and absence of mbflt-1 (190 kDa) in corneas of bottlenose dolphin (1) and Asian elephant (2). Scale bars are 200 μm.
FIG. 5 depicts a reducing western blot of Cre expression showing that 1) AC injection of NLS-Cre leads to corneal expression of Cre; 2) SC injection leads to expression in the cornea, retina, and RPE/choroid; 3) VIT injection leads to expression in lens, retina, and RPE/choroid; and 4) SR injection leads to expression in retina and RPE/choroid over 1 hr.
FIG. 6 depicts a reducing western blot of Cre expression showing that 1) AC injection of NLS-Cre leads to corneal expression of Cre; 2) SC injection leads to expression in the cornea, retina, and RPE/choroid; 3) VIT injection leads to expression in lens, retina, and RPE/choroid; and 4) SR injection leads to expression in retina and RPE/choroid over 3 hrs.
FIG. 7 depicts a reducing western blot of Cre expression showing that 1) AC injection of NLS-Cre leads to corneal expression of Cre; 2) SC injection leads to expression in the cornea, retina, and RPE/choroid; 3) VIT injection leads to expression in lens, retina, and RPE/choroid; and 4) SR injection leads to expression in retina and RPE/choroid over 8 hrs.
FIG. 8 depicts a reducing western blot of Cre expression showing that 1) AC injection of NLS-Cre leads to corneal expression of Cre; 2) SC injection leads to expression in the cornea, retina, and RPE/choroid; 3) VIT injection leads to expression in lens, retina, and RPE/choroid; and 4) SR injection leads to expression in retina and RPE/choroid over 24 hrs.
Patent Application
20070191273
