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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online December 14, 2005 as doi:10.1096/fj.05-3890fje. |
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,1
* Molecular Ophthalmology, Lions Eye Institute, Nedlands, Australia;
Centre for Ophthalmology and Visual Science, The University of Western Australia, Nedlands, Australia; and
Division of Genetics and Bioinformatics, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
1 Correspondence: Lions Eye Institute, 2 Verdun St., Nedlands, WA 6009, Australia. E-mail: binz{at}cyllene.uwa.edu.au
SPECIFIC AIMS
Laser photocoagulation (LPC) has been used to treat diabetic retinopathy with great success for many years. However, the molecular mechanism that inhibits the progression of retinal neovascularization after LPC in diabetic patients remains to be elucidated. We hypothesize that the long-term and possibly permanent modification of the gene expression pattern in the affected tissue contributes to the efficacy of LPC. We tested this hypothesis by conducting gene array analysis of lasered and unlasered mouse eyes, and examined their gene expression profiles 90 days post-LPC. Differentially expressed genes were assessed for their functional association with the process of neovascularization and LPC, as well as for the effect of the latter on the cytoarchitecture of the retina/retinal pigment epithelium (RPE) complex.
PRINCIPAL FINDINGS
1. Gene-expression pattern in the mouse eye was altered long-term after LPC
Robust multichip analysis-based processing of the data identified 107 genes as differentially expressed 90 days post-LPC, compared with unlasered control eyes (|t|>4). Thirty-four of the genes that were differentially expressed 90 days after treatment had been identified as differentially expressed at 3 days post-LPC in our earlier study. These genes therefore exhibited a true long-term change in their expression due to LPC. The majority of differentially expressed genes at 90 days post-LPC had their expression decreased by LPC according to microarray gene expression analysis with fold changes ranging from 1.5 to 2.9. Six structural genes including five crystallins (Cryaa, Cryba1, Crybb2, Crygc, Crygs) and keratin 1-12 (Krt1-12), the anti-angiogenic factor thrombospondin 1 (Tsp1), the retina and brain specific putative transcription factor tubby-like protein 1 (Tulp1) and transketolase (Tkt), a key enzyme in the pentose-phosphate pathway were all shown to be up-regulated by real-time PCR (Table 1
). Crystallin mRNA levels were induced between 4.5- and 239.7-fold compared with unlasered control tissue (qRT-PCR, Table 1
). This correlated to a significant increase in protein levels for CRY
A and CRYßB2 with fold increases of 3.7 and 3.1, respectively. The mRNA level of Krt1-12 was significantly increased in lasered eyes compared with unlasered controls, with a fold change of 41 (qRT-PCR; Table 1
). This additional expression translated into a 1.3-fold increase in detectable K12 protein within the posterior eye cup. The expression of Tsp1 was also induced both at the mRNA and protein levels (qRT-PCR fold change: 1.2; TSP1 Western blot fold change: 1.3). Another gene that exhibited a small but significant change in expression was Tulp1 (qRT-PCR fold change: 1.4). We found expression of Tkt induced after LPC (microarray fold change: 2.1; qRT-PCR fold change: 1.4).
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2. Differentially expressed genes were associated with the laser lesions
Normal retinal architecture is shown in Fig. 1
A, indicating the major components of the posterior eye cup. The RPE, photoreceptor inner and outer segments (PIS, POS), and outer nuclear layer (ONL) containing the photoreceptor cell bodies were the portions of the retina/RPE complex most affected by therapeutic argon laser treatment (Fig. 1B
). Laser spot size on delivery was 50 µm, and there was obvious proliferation of RPE cells and loss of PIS, POS, and ONL within and traction of the inner nuclear layer (INL) into the lesion core (Fig. 1B
). As the outer plexiform layer (OPL) is situated between the inner and outer nuclear layers, it stands to reason that this layer would also be affected by LPC changes to the cytoarchitecture of the retina. There appeared to be no gross morphological changes in the inner plexiform (IPL), ganglion cell (GCL) and nerve fiber layers (NFL) of these eyes (Fig. 1B
). After LPC, immunohistochemistry localized CRYA and CRYßB2 proteins to the POS along the edges of the laser lesions extending 
100 µm beyond this boundary (Fig. 1B
). TSP1-positive cells were seen again at the edges of the lesions, but in this case along the RPE cells. TULP1 staining was observed within the core of the laser lesions among the proliferating RPE cells and the glial scar, as well as the GCL. In contrast, K12 was also found in what are probably glial cells within the GCL and the NFL of lasered eyes.
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3. Differentially expressed genes were functionally associated with structural-, metabolic-, photoreceptor-, and neovascularization-related changes in the retina and RPE
A major characteristic observed in the retina/RPE/choroid tissue complex subjected to laser treatment is the loss of photoreceptors at the site of LPC, traction of photoreceptors adjacent to the laser lesions, and traction of the OPL and INL into the central area of these lesions (Fig. 1B
). Concomitantly, there is proliferation of RPE cells within these laser sites, which may be accompanied by the appearance of macrophage-like cells and glial cell infiltration. The most significant expression changes were seen in the crystallin genes at both the mRNA and protein level. There is mounting evidence that CRYA is an anti-apoptotic regulator and therefore may prevent photoreceptor and RPE cell death around the laser lesions.
Staining for the Krt1-12 protein K12 in the GCL/NFL portion of the neural retina was unexpected. However, the presence of K12 after LPC in this particular layer is significant with respect to diabetic retinopathy, as this is the site where neovascular vessels are mainly situated as they progress from the IPL to the vitreous (Fig. 1A
). It stands to reason that K12 may have a role in the control of neovascularization in addition to its known role in structural epithelial support.
Similarly to CRYA, TULP1 as a putative transcription factor may act on genes involved in neuronal cell survival, and up-regulation of this gene and its protein product may protect the remaining photoreceptors across the retina and/or stabilize those photoreceptors bordering the laser lesions by ensuring continuity of cell signaling.
Up-regulation of Tkt, a thiamine-dependent member of the pentose-phosphate pathway, has been shown to have a beneficial effect on the microvasculature in experimental diabetic animals through benfotiamine supplementation, which inhibits three major biochemical pathways implicated in the pathogenesis of hyperglycaemia-induced vascular damage. In the present study Tkt was up-regulated at the mRNA level after LPC, suggesting LPC may exert an anti-angiogenic effect through up-regulation of this gene in the retina.
Finally, TSP1 is one of the most potent anti-angiogenic factors known. Long-term up-regulation of TSP1 after LPC suggests it may induce the anti-angiogenic properties of this gene and its protein product.
CONCLUSIONS AND SIGNIFICANCE
Retinal neovascularization is a late stage secondary complication of diabetes and is the retina’s response to the development of local foci of hypoxia within this tissue complex. Localized hypoxia leads to up-regulation of vascular endothelial growth factor (VEGF) and the activation of angiogenic and endothelial cell proliferation-involved pathways. It has been demonstrated that LPC has a significant effect on the oxygenation and blood flow in the retina. This improved oxygen supply to the inner retina where neovascular vessels sprout and grow toward the vitreous (Fig. 1A
), comes directly from the choroid, which usually only supplies the outer photoreceptor layer of the retina with oxygen. The photoreceptors are very rich in mitochondria and have a very high oxygen demand. At the sites of laser lesions, photoreceptors are lost and oxygen can now travel through the glial scar of the laser lesion through to the inner retina to relieve localized hypoxia. Indeed, down-regulation of VEGF has been demonstrated repeatedly in patients with diabetic retinopathy after LPC. However, LPC does destroy a significant portion of the retina in order to preserve some vision in patients with diabetic retinopathy. While the biomechanical process of LPC is better understood, the underlying molecular mechanism has so far not been elucidated.
We know that LPC prevents continued neovascular progression and leads to regression of existing abnormal vessels. It can be argued that regression of neovascularization depends on shifting the balance from pro-angiogenic, anti-apoptotic and cell proliferation-inducing stimuli to anti-angiogenic stimuli with concomitant down-regulation of cell proliferation and differentiation-related factors. This effect may be specific to endothelial and related cell types or may more generally be associated with cell proliferation such as seen in neoplastic transformation and cancer development. Understanding which genes are affected by LPC and which may therefore have an effect on the regression of neovascularization provides the tools to develop gene based therapies for neovascular diseases, without damage to or destruction of the tissue to be treated.
In this study we have identified four potential therapeutic target genes, Tsp1, Krt1-12, Tkt, and Tulp1, which through their immunohistochemical localization and/or known or inferred functions show relevance to diabetic retinopathy and neovascularization. Up-regulation of the retina and brain-specific transcription factor Tulp1 suggests examination of other differentially expressed genes for putative Tulp1 interactions, as well as determining whether this gene has the potential to affect genes involved in angiogenic and anti-angiogenic pathways. Tkt, through its implication in diabetes-related metabolic damage to the microvasculature, makes an excellent candidate for examining its effect on vessels that have arisen through neovascularization. Localization of the K12 protein to those layers of the retina, which in the disease state are affected by neovascularization, suggests an entirely novel function for this structural protein. Finally, up-regulation of TSP1 after LPC confirms the original hypothesis that this treatment does indeed affect angiogenic factors. This is the first time long-term changes in the expression of these genes have been associated with LPC. Therefore, it suggests that modulated gene expression might contribute to the long-term inhibitory effect of LPC. These genes also present novel targets for gene-based therapies aimed at treating microangiopathies, especially diabetic retinopathy, a disease currently only treatable with LPC.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-3890fje;
This article has been cited by other articles:
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  E. Budzynski, J. H. Smith, P. Bryar, G. Birol, and R. A. Linsenmeier Effects of Photocoagulation on Intraretinal PO2 in Cat Invest. Ophthalmol. Vis. Sci., January 1, 2008; 49(1): 380 - 389. [Abstract] [Full Text] [PDF]
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