Vectorial release of matrix metalloproteinases (MMPs) from porcine RPE-choroid explants following selective retina therapy (SRT): Towards slowing the macular ageing process
Highlights
► Vectorial apical and basal MMP release can be studied using the Ussing chamber model. ► SRT increases the release of active MMP 2 on the basal side of the RPE. ► Transient vectorial MMP increase may revert age-related changes of Bruch's membrane.
Introduction
Bruch's membrane (BrM) and the retinal pigment epithelium (RPE) mediate the bi-directional transport of nutrients and waste products between the photoreceptors of the retina and the choroidal blood supply. The matrix of BrM is continuously renewed by processes of coupled synthesis and degradation, the latter pathway being mediated by a family of proteolytic enzymes called the matrix metalloproteinases (MMPs) together with their inhibitors, TIMPs. The secretion of MMPs 1, 2, 3 & 9 and TIMPs 1, 2 & 3 by RPE and choroidal endothelial cells and their presence in BrM has been demonstrated (Alexander et al., 1990; Della et al., 1996; Fariss et al., 1997; Guo et al., 1999; Hunt et al., 1993; Vranka et al., 1997). TIMP-3 has been shown to be an integral component of BrM (Kamei and Hollyfield, 1999). Additional high molecular weight MMP species (termed HMW1&2) consisting of polymers of MMPs 2&9 and a much larger macromolecular weight complex (LMMC) comprising primarily of HMW 1, HMW 2, pro-MMP 9 together with some pro-MMP 2 have also been identified in human BrM (Hussain et al., 2010a; Kumar et al., 2010).
Despite the presence of this rejuvenation mechanism, ageing BrM shows gross functional and structural deterioration. The capacity for transport of fluids, amino acids and larger molecular complexes, typified by carriers of metals, vitamins and lipids is severely curtailed (Hillenkamp et al., 2004a, 2004b; Hussain et al., 2010b; Moore et al., 1995; Moore and Clover, 2001; Starita et al., 1996). Reduced transport capability also implies impaired removal of membraneous debris extruded by the RPE and thus a vicious cycle is started with a potential to undermine the normal homeostatic support of the overlying photoreceptor layer.
Structural alterations of ageing BrM include increased thickness (Okubo et al., 1999; Ramrattan et al., 1994), the deposition of normal and abnormal extracellular matrix (ECM) material (Karwatowski et al., 1995), increased advanced glycation/lipoxidation endproducts (AGEs and ALEs) (Handa et al., 1999) which are known to be potent inhibitors of MMP activity (Mott et al., 1997; Nagai et al., 2009; Yamada et al., 2006), the accumulation of lipid-rich debris (Holz et al., 1994; Pauleikhoff et al., 1992), and accumulation of inter-molecular fibril cross-links that have been shown to reduce the susceptibility of the collagen molecule to proteolytic action (Hamlin and Kohn, 1971; Vater et al., 1979). The age-related accumulation of high molecular weight MMP species has also been suggested to sequester monomeric species thereby removing them from the activation process (Hussain et al., 2010a). Thus levels of latent MMPs 2&9 were shown to increase with age but the presence of activated forms was scarce (Guo et al., 1999). All these changes suggest a disturbance in the turnover of the ECM of BrM.
The ageing changes observed in normal donors were much more exaggerated in BrM from donors with age-related macular degeneration (AMD). Hydraulic conductivity of BrM and diffusional capacity for amino acids and carrier sized molecules was severely compromised (Hillenkamp et al., 2004a; Hussain et al., 2010b; Moore and Clover, 2001; Moore et al., 1995; Starita et al., 1996). Abnormalities in the MMP system have been implicated as levels of TIMP-3 inhibitor and pentosidine AGEs were elevated with levels of active MMPs 2&9 were significantly reduced in BrM from AMD donors compared to age-matched controls (Kamei and Hollyfield, 1999; Ishibashi et al., 1998; Hammes et al., 1999; Hussain et al., 2011). Diminished metabolic support may therefore be the initial insult that progresses to the death of RPE and photoreceptors recruiting additional inflammatory responses or choroidal neovascularization (CNV).
Stimulation of the MMP pathway could therefore constitute a viable therapeutic option for eyes with precursor stages of AMD. As such, Ahir et al., 2002 demonstrated increased hydraulic conductivity of aged BrM after cultivation with activated MMP 2 and MMP 9 isolated from cultured human RPE cells. These authors also showed that proliferating RPE cells release copious amounts of activated MMPs. It can thus be hypothesized that the resolution of drusen after conventional laser (Ho et al., 1999) or diode laser (Rodanant et al., 2002) treatment of RPE observed in clinical trials was caused by secretion of ECM-degrading enzymes by migrating and proliferating RPE cells during closure of the laser wound. However, both conventional continuous wave lasers (Roider et al., 1993a) and low energy diode lasers (Mojana et al., 2011), although targeting RPE, also cause irreversible collateral thermal damage to the outer retina. Therefore, in the clinical trials, laser treatment was applied only once, the number of laser spots was kept to a minimum and spots were delivered into the peripheral macula to avoid undue damage of the central macula (Ho et al., 1999; Rodanant et al., 2002; Owens et al., 2006; Maguire et al., 2006). Hence, improvement of visual function may be expected to be limited (Ho et al., 1999; Friberg et al., 2009) or absent (Owens et al., 2006).
Selective Retina Therapy (SRT) is a micropulse laser technique that selectively targets the RPE but spares the neurosensory retina without causing microscotoma (Brinkmann et al., 2006; Roider et al., 2000, 1999, 1993b, 1992). Selective damage of the RPE is achieved by formation and rapid expansion of microbubbles around the melanosomes in the RPE cells leading to disruption of the cell structure without thermal transients spreading into the adjacent tissue (Brinkmann et al., 2000). This confinement of heat flow to the interior of a given RPE cell ensures that the overlying photoreceptors and the underlying BrM with the choroid are spared from damage. The mechanism of SRT could enable the positive features of previous treatments for early AMD with conventional lasers to be harnessed without entertaining the negative effects such as the promotion of CNV or microscotoma in the central visual field.
The present investigation was designed to assess the potential use of the SRT technique for inducing the release of activated MMPs from RPE cells as a vehicle to address the pathological alterations of BrM associated with AMD. An explant culture system was initiated to allow the determination of RPE apical and basal compartment MMP release patterns and to assess the effect of the SRT laser using energy levels below and above the threshold of RPE cell disruption.
Section snippets
Porcine RPE-choroid preparation
Fresh porcine eyes were obtained from a local abattoir and experiments were initiated within 3–4 h of death. Whole globes were briefly immersed in antiseptic solution. A circumferential incision at the pars plana allowed the removal of the anterior segment and vitreous followed by gentle peeling of the retina. The remaining globe was cut into quadrants and the RPE-BrM-choroid layer gently separated from the sclera using forceps and scissors. A suitably sized RPE-choroid preparation was then
RPE-choroid organ culture
Calcein-AM staining confirmed the structural integrity of the monolayer with preservation of morphological characteristics of the RPE cells over an examined incubation period of 6–7 days (Fig. 2a,b). Cell viability remained intact over this period under restricted nutritional conditions with 1% porcine serum supplement in the medium. Paraffin embedded cross-sections of three RPE-choroid preparations showed polarized RPE cells with basal nuclei and apical pigment granules after 24 h of
Discussion
As opposed to previously used organ culture (Del Priore et al., 1989; Flaxel et al., 2007; Miura et al., 2010) and cell culture models (Ahir et al., 2002), the newly established modified Ussing-chamber system of the present study has allowed an assessment of the differential release profile of MMPs from the apical and basal aspects of an intact RPE monolayer. In both control and lasered preparations, the release of pro-MMPs 2&9 occurred primarily into the basal compartment. Furthermore, very
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