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First published online 17 July 2006
doi: 10.1242/jcs.03059

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JUN siRNA regulates matrix metalloproteinase-2 expression, microvascular endothelial growth and retinal neovascularisation

¹ Centre for Vascular Research, The University of New South Wales and Department of Haematology, The Prince of Wales Hospital
² Inflammatory Diseases Research Unit, University of New South Wales, Sydney NSW 2052, Australia

View larger version (33K): [in a new window]   Fig. 1. Jun siRNA suppresses JUN and MMP-2 protein expression in murine microvascular endothelial cells. (A) Growth-quiescent bEND-3 cells transfected with 0.1 µM (thin bar) or 0.4 µM (thick bar) of Jun siRNA, siRNAscr or mock-transfected and incubated in medium containing serum for 2 hours before western blot analysis (for JUN or ß-actin) of whole cell extracts. (B) Growth-quiescent bEND-3 cells transfected with 15 µg of pGL3-prom bearing two JUN/AP-1 binding sites and 0.1 µM (thin bar) or 0.4 µM (thick bar) of Jun siRNA, siRNAscr, Egr-1 siRNAscr or mock transfected were incubated in medium containing serum for 24 hours before determination of luciferase activity in the cell lysates. (C) RT-PCR analysis for Jun using extracts of growth-quiescent bEND-3 cells transfected with 0.1 µM (thin bar) or 0.4 µM (thick bar) of Jun siRNA, siRNAscr, Egr-1 siRNAscr or mock-transfected and incubated in medium containing serum for 2 hours. (D) RT-PCR analysis for MMP-2 using extracts of growth-quiescent bEND-3 cells transfected with 0.1 µM (thin bar) or 0.4 µM (thick bar) of Jun siRNA, siRNAscr, Egr-1 siRNAscr or mock transfected and incubated in serum-free medium containing 10 ng/ml TGFß for 24 hours. (E) Gelatin zymography demonstrating MMP-2 bioactivity in supernatants of transfected bEND-3 cells. The cells were transfected with 0.1 µM (thin bar) or 0.4 µM (thick bar) of Jun siRNA or siRNAscr and incubated in serum-free medium containing 10 ng/ml TGFß for 24 hours. Data are representative of at least two independent experiments performed in duplicate or triplicate and are mean values ± s.e.m. *P<0.05 compared with no siRNA control by Student's t-test.

View larger version (13K): [in a new window]   Fig. 2. Jun siRNA inhibits growth of murine microvascular endothelial cells. Growth-quiescent bEND-3 cells transfected with 0.02 µM (thin bar) or 0.05 µM (thick bar) of Jun siRNA, siRNAscr, siRNA2, siRNA2scr or mock transfected were incubated in medium containing serum for 48 hours before trypsinisation and quantification of cell numbers using a Coulter counter. The data are representative of at least two independent experiments performed in triplicate. *P<0.05 compared with the no siRNA control by Student's t-test.

View larger version (88K): [in a new window]   Fig. 3. Jun siRNA inhibits murine microvascular endothelial cell regrowth from the wound edge. bEND-3 cells were transfected with 0.1 or 0.4 µM of Jun siRNA, siRNAscr or mock transfected (No siRNA). Injury was performed by scraping the monolayer, leaving the cells in medium containing serum for 48 hours before fixation, Haematoxylin-Eosin staining and (A) microscopy (200x) or (B) quantification of cell numbers in the denuded zone (0.1 µM, thin bar; 0.4 µM, thick bar). The data are representative of at least two independent experiments performed in triplicate. *P<0.05 compared with the no siRNA control by Student's t-test.

View larger version (67K): [in a new window]   Fig. 4. Jun siRNA inhibits murine microvascular endothelial tubule formation. bEND-3 cells transfected with 0.1 µM of Jun siRNA, siRNAscr or mock transfected were seeded onto Matrigel-coated wells and spontaneous tubule formation was quantified by counting tubules using phase-contrast microscopy (under 100x magnification) at the times indicated. The data is representative of at least two independent experiments performed in triplicate. The figure shows representative photomicrographs after 6 hours at 200x magnification. *P<0.05 compared with control by Student's t-test.

View larger version (58K): [in a new window]   Fig. 5. Jun siRNA inhibits angiogenesis in mice. C57BL/6 mice were implanted subcutaneously with Matrigel plugs containing 0.5 µg FGF-2 and 100 µg Jun siRNA or Jun siRNAscr or no siRNA. Blood vessels (CD31+, top panel inset) in the cross-sectioned plugs were quantified by light microscopy 14 days after implantation. The figure shows representative cross-sections of the treated plugs (magnification, 400x). *P<0.05 by Student's t-test between groups as indicated.

View larger version (63K): [in a new window]   Fig. 6. siRNA targeting Jun inhibits neovascularisation in mice following hyperoxia-induced proliferative retinopathy. Postnatal day 6 (P6) C57BL/6 mice were exposed to hyperoxia (75% oxygen) for 4 days. P10 mice were returned to normoxia, and a bolus intravitreal injection of 20 µg of either Jun siRNA or siRNAscr was administered. Where indicated the siRNA was co-administered with 0.08 µg TGFß with or without 20 µM cis-9-octadecenoyl-N-hydroxylamide (cis-9-o-N-h). Mice were left at room oxygen for a further 7 days before P17 pup eyes were enucleated and fixed in 10% formalin in PBS. (A) Serial cross-sections of the eyes were stained with Haematoxylin and Eosin and blood vessels in the retina were quantified by light microscopy under 400x magnification and expressed as the mean ± s.e.m. Representative images at 100x magnification (upper panels). Jun siRNA inhibits hyperoxia-induced neovascularisation (lower panel). *P<0.05 by Student's t-test between groups as indicated. Immunoreactivity for (B) JUN, (C) MMP-2 or (D) Sp1 in retinal microvessels of mice treated intravitreally (20 µg) by single administration of Jun siRNA or siRNAscr or vehicle (no siRNA). Western blot analysis in C and D was performed with bEND-3 lysates as described in the Materials and Methods. Arrows in B-D indicate specific immunostaining (magnification, 1000x).