|
RESULTS
Results of FFA At 1 day and 3 days after photocoagulation, obvious fluorescence leakage could not be detected. At 1 week, 2 weeks and 4 weeks after laser treatment, fluorescence leakage could be seen at many sites of photocoagulation scars, along with the time, the areas of fluorescence leakage expanded and its intensity increased, which proved the development of CNV (Figure 1).
Expression of MMP9 and TIMP3 mRNA In normal mouse, only a few expressions of MMP9 and TIMP3 mRNA could be seen in choroids and areas near the retinal pigment epithelium (RPE). At 1 day after laser photocoagulation, local elevation of retina and breakdown of integrity of RPE could be observed at sites of photocoagulation, wherein with a small quantity of signals of MMP9 and TIMP3 mRNA. At 3 days after laser photocoagulation, the pigment proliferated and became confused. The expression of MMP9 and TIMP3 mRNA increased with a different degree. At 1 week after laser photocoagulation, CNV developed and last for at least 4 weeks after photocoagulation. During this period, pigment was still confused, and spindleshaped RPE cells covering the CNV tissues could also be seen. Both expression of MMP9 and TIMP3 mRNA were obviously in the areas of CNV and surrounding tissues (Figure 2).
Figure 1 Photograph of FFA 1 week after photocoagulation At 1 week after laser photocoagulation, fluorescence leakage could be seen at the site of photocoagulation(略)
After image analysis using the KS 400 image analysis software package, the positive ratio were calculated and showed in Table 1 and Figure 3.
As a result, both expression of MMP9 and TIMP3 mRNA had a dynamic changes during the development of CNV. They all increased significantly in the early stage after laser treatment, and then stabilized at a high level. For MMP9 mRNA, the expression was 1, 2, 4wk>3d>1d (P<0.05), whereas TIMP3 mRNA, 3d, 1, 2, 4wk>1d (P<0.05). Namely, the expression of TIMP3 mRNA became stable at 3 days after photocoagulation, whereas MMP9 mRNA increased constantly until 1 week after laser treatment.
DISCUSSION
MMPs are a group of extracellular enzymes that share a number of common characteristics, such as the presence of zinc and a conserved amino acid sequence at their active sites. They are a highly regulated family of at least 14 structurally related enzymes capable of degrading most, if not all, of the components of the ECM.
The MMPs are grouped into collagenases (MMP1, 8, 13), gelatinases (MMP2, 9), stromelysins (MMP3, 7, 10, 11, 12), and membranetype matrix metalloproteinase (MMP14, 15, 16, 17). These enzymes and their inhibitors, TIMPs, Figure 2 Photography of in situ hybridization in CNV tissues are involved in maintaining the dynamic equilibrium of ECM.
A: negative control; B: expression of MMP9 mRNA (1 week after laser photocoagulation); C: expression of TIMP3 mRNA (1 week after laser photocoagulation) original magnification×200
Figure 3 The changes of expression of MMP9 and TIMP3 mRNA during the development of CNV(略)
Several ocular tissues secrete MMPs and TIMPs in culture, including cornea, trabecular meshwork and RPE. MMPs have also been found in aqueous humor, tears and sclera. MMP9, the 92kDa gelatinase B, is present in human Bruchs membrane, vitreous and interphotoreceptor matrix (IPM)[13].
In order to investigate the possible role of MMPs in CNV, Steen et al[4] analyzed the mRNA expression of MMPs in subfoveal fibrovascular membranes from five patients with agerelated macular degeneration. They found that MMP9 expression was distinctly expressed by cells at the margins of the membranes and often in proximity to a thickened Bruchs membranelike layer under the retinal pigment epithelial cells. It may be an attempt by the RPE cells to reform a Bruchs membranelike structure around the CNV lesion in an effort to create a physical barrier that inhibits further CNV growth. The presence of MMP9expressing cells next to this Bruchslike layer possibly heralds a processing collagen IV degradation so that the CNV membrane can grow further into the subretinal space. Das et al[5] also observed that levels of MMPs (MMP2 and MMP9) in retinas were significantly increased in an animal model with ischemiainduced retinal neovascularization, namely, newborn mice exposed to the variable oxygen cycle. Neovascularization was significantly inhibited (72% reduction) with intraperitoneal administration of a synthetic MMP inhibitor, BB94 (1mg/kg).
In this study, we found that there was some expression of MMP9 mRNA in retina of C57BL/6J mouse during the early period after laser photocoagulation. Then the expression increased along with the time until to the CNV development at 1 week after laser treatment. As a follow, it would stabilize at a high level. According to these findings, combinating with references showed above, we presume that MMP9 may play a role in the pathogenesis of retinal neovasculization and CNV by degrading of ECM.
TIMPs are a family of MMP inhibitors thought to act as local regulators of matrix degradation by the MMPs. Recent histological studies have examined the location and expression of the TIMPs in retinal and choroidal tissue. While TIMP1 could not be found, TIMP2 was shown to be present in Bruchs membrane and choroid, and TIMP3 mRNA expression was localized to the RPE and choroidal endothelial cells, and there is general consensus that TIMP3 in Bruchs membrane is synthesized and secreted by the RPE. TIMP3 is unique in having a strong affinity for ECM, while the other two are found predominantly in the media from cultured cells [6]. It is suggested that TIMP3 normally functions for maintenance of the ECM in Bruchs membrane [7].
One role of TIMP3 in Bruchs membrane may be as a potent local inhibitor of MMP activity, regulating the rate of Bruch's membrane turnover, as well as limiting choroidal neovascularization. In 2000, Takahashi et al[8] injected hemagglutinating virus of Japan liposomes containing hemagglutin epitopetagged TIMP3 gene into the subretinal space in rat eyes. Three days after transfection of TIMP3 gene into retinal pigment epithelium cells, intense laser photocoagulation was performed and the incidence of CNV was assessed by FFA. They found that exogenous TIMP3 mRNA expression in the choroid and retina was detected on day 3. The efficiency of TIMP3 gene transfection into retinal pigment epithelium cells was greatest on day 7 and decreased gradually thereafter. The incidence of CNV in TIMP3 genetransfected eyes was markedly decreased compared with controls (15% vs 75%). This study shows that TIMP3 gene can be transferred into rat retinal pigment epithelium and that TIMP3 gene overexpression can inhibit development of experimental CNV. This method may represent a future treatment modality for human macular degeneration associated with CNV. Murata also confirmed the possibility of gene therapy (coding for TIMP) for the treatment of CNV [9].
上一页 [1] [2] [3] 下一页 |
