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L. K. Buie, M. G. Spiga, K. D. Kennedy, W. C. Fowler, T. Borras; Overexpression of MMP1 by an Adenoviral Vector (Ad) Hampers Myocilin (MYOC) Expression. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3222.
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© ARVO (1962-2015); The Authors (2016-present)
Recently we have shown that steroid-induced ocular hypertension in sheep is significantly lowered by an intracameral injection with an Ad vector containing a GRE-inducible MMP1 cDNA (AdhGRE.MMP1). In this study we investigated mechanisms responsible for the pressure reduction, and determined the effects of overexpressing MMP1 on MYOC production. For this, we developed a stationary TM organ culture (STM) using human corneal rims from surgical transplants.
Primary human trabecular meshwork cells (HTM) and perfused organ cultures were infected with AdGRE.MMP1 in the presence of 0.1 µM DEX. For GRE competition experiments, HTM cells were nucleofector-transfected with pMG12 (same cassette as AdGRE.MMP1) and pGRE.Luc. Residual corneal rims were cut in wedges and placed in 1 ml of serum-free media in 24-wells overnight. Media was removed and 40 µl of AdhGRE.MMP1, AdCMV.GFP, or vehicle were laid on the TM of the wet tissue. After 1 h, SFM media was added, replaced with serum-containing medium at 6-16 h and incubated for 3-4 days. TM was dissected for RNA and one wedge embedded for fluorescent histology. Expression of MMP1 and MYOC was determined by TaqMan PCR normalized to 18S. Secreted MYOC was evaluated by WB, and secreted MMP1 by WB and ELISA.
Adh.GREMMP1 infection of cells and perfused organ cultures in the presence of DEX increased secreted MMP1 and extensively reduced secreted MYOC levels induced by DEX alone (WB). Expression of MYOC in the Adh.GREMMP1 infected cells was reduced 15-fold (TaqMan). Nucleofector-transfected cells with either pMG12 or pGRE.Luc in the presence of DEX, reduced MYOC cDNA by 2-14 -fold, indicating a competition with an active GRE MYOC promoter element. However, STM organ cultures, expressing high levels of MYOC in the absence of DEX, showed also reduced MYOC expression (2-5-fold) in the Adh.GREMMP1 infected TM. Fluorescence microscopy showed GFP transduction in the TM.
Overexpression of steroid-inducible MMP1 in the presence/absence of DEX downregulates expression of MYOC. STM organ cultures are a good source for Ad gene transfer TM studies. The IOP lowering of steroid-induced hypertension in sheep by AdhGRE.MMMP1 might be enhanced by a modulation of MYOC production.
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