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Oscar Alcazar, Scott W. Cousins, Maria E. Marin-Castaño; MMP-14 and TIMP-2 Overexpression Protects against Hydroquinone-Induced Oxidant Injury in RPE: Implications for Extracellular Matrix Turnover. Invest. Ophthalmol. Vis. Sci. 2007;48(12):5662-5670. doi: 10.1167/iovs.07-0392.
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purpose. To investigate whether overexpression of MMP-14 and/or TIMP-2 would overcome the effect of nonlethal oxidant injury with hydroquinone (HQ) on MMP-2 activity.
methods. Human MMP-14 and TIMP2 cDNA were cloned into a mammalian expression vector. Transient transfections were performed on human ARPE-19 cells. The cells were incubated 48 hours after transfection with a nonlethal dose of HQ for either 6 or 18 hours and then were collected for protein determination or RNA isolation. MMP-2 protein and activity were determined by Western blot and zymography. The extracellular matrix (ECM) components type I and type IV collagen and laminin were analyzed by Western blot analysis and real-time PCR.
results. HQ for 6 hours modestly decreased MMP-2. MMP-2 recovered only after co-overexpression of MMP-14 and TIMP-2, but activity further decreased after HQ for 18 hours. MMP-14 or TIMP-2 overexpression alone contributed as much as the co-overexpression to the recovery of MMP-2 activity. MMP-2 protein seemed not to be altered. Type I collagen and laminin transcriptional levels remained unaffected, whereas type IV collagen transcripts decreased with HQ. Transfection with MMP-14 and/or TIMP-2 contributed to the return of type IV collagen levels to normal. On the other hand, type I and IV collagens and laminin protein accumulated after HQ treatment, an effect prevented by transfection.
conclusions. MMP-14 and TIMP2 contribute to the maintenance of adequate levels of MMP-2 activity in ARPE-19 cells after oxidant injury. In addition, changes in ECM components may result as a consequence of MMP-2 activity and may be relevant to the progression of dry AMD.
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