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Purpose: The purpose of this study was to determine the endogenous regulation pattern of tissue inhibitor of metalloproteinase-2 (TIMP-2) in the tree shrew sclera during myopia development and investigate the capacity of exogenous TIMP-2 to inhibit matrix metalloproteinase-2 (MMP-2) in vitro and both scleral collagen degradation and myopia development in vivo., Methods: TIMP-2 expression in the sclera during myopia development was assessed using polymerase chain reaction. In vitro TIMP-2 inhibition of MMP-2 was investigated using a gelatinase activity plate assay and zymography. Tree shrews were injected with a collagen precursor before undergoing monocular form deprivation and concurrent daily subconjunctival injections of either TIMP-2 or vehicle to the form-deprived eye. In vivo ocular biometry changes were monitored, and scleral tissue was collected after 12 days and assayed for collagen degradation., Results: The development of myopia was associated with a mean reduction in TIMP-2 mRNA expression after 5 days of form deprivation (P < 0.01). Both activation and activity of MMP-2 were inhibited by TIMP-2 with an IC50 of 10 to 20 and 2 nM, respectively. In vivo exogenous addition of TIMP-2 significantly reduced myopia development (P < 0.01), due to reduced vitreous chamber elongation (P < 0.01). In vivo TIMP-2 treatment also significantly inhibited posterior scleral collagen degradation relative to vehicle-treated eyes (P < 0.01), with levels similar to those in control eyes., Conclusions: Myopia development in mammals is associated with reduced expression of TIMP-2, which contributes to increased degradative activity in the sclera. It follows that replenishment of this TIMP-2 significantly reduced the rate of both scleral collagen degradation and myopia development.

Purpose: Matrix metalloproteinases (MMPs) and their inhibitors play a role in the pathobiology of retinal detachment (RD) and proliferative vitreoretinopathy (PVR). Proliferative vitreoretinopathy is facilitated by chronic retinal detachment and involves excessive deposition of extracellular matrix (ECM) proteins. Matrix metalloproteinase-2 and -13 are important modulators of the ECM which have not been evaluated in RD. The purpose of this study was to investigate the retinal expression of select MMPs, including MMP-12, MMP-13, and associated inhibitors in a murine model of retinal detachment., Methods: Transient or chronic retinal detachments (RDs) were induced by subretinal injection of either saline (SA) or hyaluronic acid (HA) in C57BL/6 mice. To confirm that the HA-RD model has features consistent with PVR-like changes, glial activation and subretinal fibrosis were evaluated with immunofluorescence, dilated fundus examination, and spectral-domain optical coherence tomography (SD-OCT). Gene expression was quantified by qRT-PCR. Proteins were assayed by immunoblot and immunohistochemistry., Results: Hyaluronic acid RD eyes developed gliosis and subretinal fibrosis on dilated exam, SD-OCT, and immunofluorescence analysis. Gene expression of Mmp-12 and Mmp-13, and Timp-1 was strongly upregulated at all time points in RD compared with controls. Timp-2, Mmp-2, and Mmp-9 expression was modest. Hyaluronic acid RDs exhibited more MMP and TIMP expression than SA-RDs. MMP-12, -13, and TIMP-1 proteins were elevated in RDs compared with controls. Immunohistochemistry revealed moderate to strong MMP-13 levels in subretinal space macrophages., Conclusions: Fibrosis can develop in the HA-RD model. There is an upregulation of select MMPs that may modulate the wound healing process following RD.

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.

Purpose: Matrix metalloproteinases (MMPs) are involved in trabecular meshwork (TM) extracellular matrix metabolism and have been shown to increase aqueous outflow facility. The purpose of this study was to characterize effects of cytokines, a phorbol ester, and prostanoids on the expression of MMP-1, -2, -3, and -9 and tissue inhibitors of metalloproteinases (TIMP)-1 and -2 in cultured human TM cells., Methods: Five human TM cell strains were treated with selected compounds. Levels of proMMPs and TIMPs in cell media were quantified by ELISA. MMP-3 activity was assayed by casein zymography., Results: All human TM cell strains produced detectable basal amounts of proMMPs and TIMPs. 12-O-tetradecanoyl-phorbol-13-acetate was effective in increasing the levels of proMMP-1, -3, and -9 and TIMP-1. Its effect on proMMP-1 was concentration-dependent with an EC(50) of 2 to 3 nM. Interleukin (IL)-1alpha did not affect levels of proMMP-1 and -2 or the TIMPs, but was most efficacious in increasing proMMP-3 production with an EC(50) of 0.5 ng/mL. The IL-1alpha-induced upregulation of proMMP-3 correlated with an increase in MMP-3 activity. Tumor necrosis factor-alpha activated proMMP-3 production in some but not all cell strains. Platelet-derived growth factor-BB was generally ineffective in modulating MMP and TIMP levels. Prostaglandins E(2) and F(2alpha) at 10 micro M did not affect levels of proMMP-1 or -3., Conclusions: The expression of the different MMPs and TIMPs in human TM cells was independently regulated. Production of MMP-3 was maximally activated by IL-1alpha. The IL-1alpha-stimulated expression of MMP-3 provides a probable mechanism for IL-1alpha-enhanced aqueous outflow.

Purpose: To determine the effect of treatment with latanoprost on the tissue inhibitors of metalloproteinase (TIMP)-1 and -2 in cultured human ciliary muscle (HCM) cells., Methods: Confluent serum-starved HCM cells were exposed to increasing concentrations of latanoprost acid (LA, 1 nM to 10 micro M) for 6, 18, and 24 hours. TIMP-1 and -2 mRNA transcripts were evaluated by RT-PCR. Gelatin zymography was used to measure changes in the amount of matrix metalloproteinase (MMP) in the culture medium. To evaluate the potential role of PKC, HCM cells were treated with phorbol 12-myrisate 13-acetate (PMA) in the absence or presence of the PKC inhibitor bisindolylmaleimide I (Bis I) or the PKA inhibitor KT5720. Data were quantitated by densitometry and statistically analyzed with the Student-Newman-Keuls test., Results: TIMP-1 and -2 mRNA transcripts and proteins were detected in primary cultures of HCM cells. TIMP-1 mRNA levels were unchanged at 6 hours, but increased 45% +/- 17% and 54% +/- 13% in cultures exposed for 18 hours to 1 and 10 micro M LA, respectively (n = 3). In contrast, 6 hours of exposure to LA increased expression of TIMP-2 mRNA by up to 11.3% +/- 0.2% (n = 3). However, no significant induction of TIMP-2 mRNA was observed at either 18 or 24 hours (n = 3). TIMP-1 protein was significantly increased in cultures exposed to LA for 18 and 24 hours. In contrast, TIMP-2 protein expression was insignificantly different from control cultures at 6, 18, and 24 hours of treatment. HCM cells exposed to PMA for 24 hours produced similar increases in TIMP-1 mRNA levels, as seen with latanoprost (n = 5). However, no significant induction of TIMP-2 mRNA was observed. Zymographic analysis of the media from these cultures confirmed dose-dependent increases of MMP-1 at 6, 18, and 24 hours, whereas dose-dependent increases in MMP-2 were seen only after 24 hours' exposure to LA (n = 3). TIMP-1 protein levels were increased 27% +/- 9.3% and 15% +/- 11% in the media of cells exposed for 24 hours to 100 nM LA and 100 nM PMA, respectively (n = 5). The increases in TIMP-1 protein induced by LA were essentially eliminated by Bis I (n = 3) and unaffected by KT5720 (n = 3)., Conclusions: For the most part, TIMP-1, and not TIMP-2, contributes to regulation of MMP within the uveoscleral outflow pathway after exposure to latanoprost. Moreover, this induction appears to be meditated by activation of PKC.

Purpose: Myopia, or nearsightedness, is characterized by excessive lengthening of the ocular globe and is associated with extracellular matrix remodeling in the posterior sclera. The activity of gelatinase A, a member of the matrix metalloproteinase family, has been shown to increase in the posterior sclera during the development of induced myopia in several species. In the present study, the distribution and relative expression of gelatinase A and its associated inhibitor, tissue inhibitor of metalloproteinases (TIMP)-2, were measured within the fibrous scleras of experimentally myopic (form-deprived) eyes, control eyes, and eyes recovering from form deprivation to better understand the mechanisms that regulate scleral remodeling and the rate of ocular elongation., Methods: Total RNA was extracted from the posterior scleras of form-deprived chick eyes, eyes recovering from deprivation myopia, and paired contralateral control eyes, and subjected to northern blot analysis analyses using cDNA probes to chicken gelatinase A and TIMP-2. The distribution of gelatinase A and TIMP-2 mRNAs was evaluated by in situ hybridization on frozen sections of chick scleras using 33P-labeled RNA probes. Gelatinase A activity within the fibrous scleras of form-deprived eyes and paired contralateral recovering eyes was evaluated by gelatin zymography., Results: Northern blot analysis indicated that the relative expression of gelatinase A was increased by 128% in deprived eyes (P = 0.009), whereas after 1 day of recovery, levels were decreased by 80% in scleras from recovering eyes (P = 0.005). In contrast, TIMP-2 expression was significantly decreased (-53%, P = 0.027) in the posterior scleras of form-deprived eyes. No significant differences were detected in levels of TIMP-2 expression between recovering eyes and paired control eyes. In situ hybridization indicated that most of the gelatinase A transcripts were present in the fibrous layer of the posterior scleras from form-deprived and recovering eyes., Conclusions: Changes in the steady state levels of gelatinase A and TIMP-2 mRNA lead to changes in gelatinase activity within the fibrous sclera and mediate, at least in part, the process of visually regulated ocular growth and scleral remodeling.