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Retinal Cells Apoptosis and the Correlation with CTGF The expression of apoptosis was examined by Tunel staining and retina samples were processed from control, DM4W, DM8W, DM16W, DM24W and DM16W interfered with CTGFsiRNA groups. DM16W was also the noninterfered group compared with interfered with CTGFsiRNA group. The TUNELpositive nuclei were identified by a brown reaction product and were found in all regions of the retina. After only 4 weeks, diabetic retinas had seldom TUNELpositive nuclei in ganglion cell layer compared to the control. At 8 weeks, the diabetic retinas had more TUNELpositive nuclei than the control(Figure 2).The cell of apoptosis count is 12.6 cells/mm2 and the positive stained included ganglion cells and the glial cell.
appeared in diabetic retina a:normal retina; b: diabetic 4 weeks; c: diabetic 8 weeks; d: diabetic 16 weeks; e: diabetic 24 weeks; f: diabetic 16 weeks interfereced by CTGFsiRNA. The internal limiting membrane (ILM), the ganglion cell layer (GCL), the inner plexiform layer (IPL), the outer plexiform layer (OPL), and the outer nuclear layer (ONL).The difference is significant(aP<0.05,bP<0.01).
At 24 weeks of diabetes TUNELpositive nuclei localized in all regions of the retina, including vascular endothelial cells and the cells in inner nuclear layer, and the cell apoptosis counts increased to 25.8cells/mm2. There is a downregulation of apoptosis in interfered retina compared with uninterfered retina. The CTGFsiRNA protects the retina from apoptosis, compared with the uninterfered retina, in which the difference is significant (P<0.01). Next we tested for a relationship between the apoptosis and CTGF mRNA in retina. As evidence above, the correlations between the apoptosis expression and CTGF expression (r=0.871, P=0.011, Figure 3). There was a significant correlation between the apoptosis and the expressions of CTGF in diabetic retina.
DISCUSSION
The present study indicates there is an increased expression of the CTGF gene level and the apoptosis of early diabetes and the degree of increasing became stronger with the diabetic development. The diabetic retinas showed apoptosis in ganglion cells layer chiefly. We can come to a conclusion that this change is a very early marker of diabetesinduced retinal changes and it occurred before the onset of visible vascular lesions. Then we used siRNA targeting with CTGF to silence the CTGF gene, which is a valuable tool for investigating the function of gene products in tissues[1618]. CTGFsiRNA could effectively downregulate the expression of CTGF in diabetic rat retinas, and a significant inhibition of apoptosis in the retina occurred by the interfered with CTGFsiRNA. There was a strong correlation between apoptosis and CTGF in diabetic rat retinas. So we demonstrated CTGF might be affected on the apoptosis in diabetic retina of rat.
Increased apoptosis is implicated in several other diabetic complications such as neuronial apoptosis in neuropathy, cardiomyocyte apoptosis in cardiomyopathy, and mesangial cell apoptosis in nephropathy. Diabetes can affect capillaries[19], neurons, and glia[19] within the retina and alters the function and structure of all retinal cell types. Much evidence had shown the apoptosis in the ganglion cells layer, the inner nuclear layers[20], and photoreceptors and microvascular cells[19] in diabetic retina. Direct diabetes damage to glial cell or neuronal metabolism would directly impact neurotransmission[21] and may lead to apoptosis of retinal neurons and visual field defects. Indeed, retinal axons are lost before the onset of visible vascular lesions[22]. Recent reports also demonstrate that impaired local responses on the multifocal electroretinograms predict subsequent development of vascular lesions[23]. Vision depends on neuronal function, so most forms of vision impairment with clear ocular media must include neuronal dysfunction definitely. Further work is needed to determine how alterations in ganglion, glial, microglial, and neuronal cell interactions reduce the quality of vision.
CTGF is a cysteinerich matricellular protein belonging to the CCN family of proteins, which have many diverse functions such as angiogenesis, fibrosis and apoptosis and so on. Recently, various studies have shown that CTGF expression at the mRNA or protein level in retina has previously been demonstrated in vivo in diabetic rat[24] and human[25], as well as in cultured retinal microvascular cells[26]. CTGF has been shown to be upregulated in the retina together with endothelial cell death. These are believed to be the result of metabolic changes caused by hyperglycemia and advanced glycation end products (AGEs)[27]. Overexpression of CTGF in cultured human aortic smooth muscle cells, a cell type closely related to pericytes and mesangial cells induces apoptosis by activating caspase 3[28]. Moreover, the involvement of Cyr61 and CTGF in pericyte detachment and anoikis was implicated in the pathogenesis of DR[29]. Cyr61 and CTGFinduced apoptosis was mediated through the intrinsic pathway and involved the expression of genes that have been functionally grouped as p53 target genes. Expression of the matrix metalloproteinase2 gene, a known target of p53, was increased in pericytes overexpressing either Cyr61 or CTGF. Inhibition of matrix metalloproteinase2 had, at least in part, a protective effect against Cyr61 and CTGFinduced apoptosis.
Thus, it is possible that upregulation of CTGF may contribute to inducing apoptosis, especially in the vascular endothelium cells, ganglion cells, perhaps included the glial cells. This upregulation leads to the loss of retinal cells as a critical early event. And then the vascular cells affected suggest that cell loss may be a direct result of a consequence of widespread vascular disease in the etiology of diabetic retinopathy[30]. But Alistair J et al indicated that most of the apoptotic cells in the retina are not endothelial cells or pericytes. Judging from such different findings, there might be an unidentified mechanism modulating the apoptosis in the diabetic retina.
Together, these studies leave little doubt that apoptosis is the earliest detectable changes in diabetes. Regardless of whether the initial events begin in blood vessels or neural cells, the clinical stage of diabetic retinopathy manifest cellular, histological, and functional features of a retinal neuropathy[31]. To the best of our knowledge, there is no evidence that a primary, selective defect in vascular cells is sufficient to cause diabetic retinopathy. Clearly, it is essential to treat both the vascular and neural elements of the retina to preserve vision. This concept permits a new paradigm for understanding the mechanism of vision impairment in diabetes and provides therapeutic targets that are directly linked to vision[32,33]. In summary, this study suggests that CTGF may be involved in apoptosis which is a characteristic of early diabetic retina. siRNA targeting CTGF seems to have the advantage of ameliorating retina apoptosis directly or indirectly. This study provides evidence treatment strategies that intravitreous injection of siRNA containing potentially therapeutic transgenes treatment[34,35]. However, it remains unclear how upregulated expression of CTGF in the diabetic retina and the exact mechanism leading to apoptosis in STZ rats should be further investigated. Meanwhile, we must be cautious in interpreting these findings, because animal models of diabetic retinopathy do not exhibit advanced retinal lesions such as those seen in the man.
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