DNA microarrays are powerful tools that allow for genome-wide gene expression profiling of cells or tissues.
32 In this study, we used a cDNA array testing for approximately 10,000 genes to identify modification of the gene expression profile of pooled ocular posterior segments in ovariectomized mice rescued or not by substitutive E2 therapy. In this experimental setting, less than 1% of the genes tested showed noticeable expression changes. Among them, the YKL-40 gene showed the largest expression change, with lower values in OVX E2-treated mice. This observation was confirmed by semiquantitative RT-PCR in a second set of experiments performed on individual mice tissues. YKL-40 gene expression was further studied in normal and pathologic human retinas.
YKL-40 expression has been reported in human RPE, with serial analysis of gene expression technology (SAGE).
33 In the current study, YKL-40 was consistently expressed in normal human eyes both in neural retina and in the RPE-choroid complex. High concentrations of YKL-40 in serum correlates with morbidity in different human diseases such as active rheumatoid arthritis and hepatic fibrosis, as well as with death of recurrent colorectal cancer.
34 35 36 YKL-40 is produced by monocytes in the media of arteritic vessels, in inflamed synovial membranes, and in atherosclerotic plaques, suggesting a role for YKL-40 in tissue remodeling.
37 38 In vitro, YKL-40 is one of the most abundant proteins secreted by cultured chondrocytes.
39 The regulation of YKL-40 expression, however, is largely unknown. In this study YKL-40 was significantly downregulated by estrogen supplementation in the ocular posterior segment of OVX mice. Furthermore, YKL-40 expression was upregulated both in pathologic human and experimental CNV, although we cannot exclude bias caused by our selecting specimens from patients for whom standard laser therapy could not be used. Taken together with the migratory properties of YKL-40 in endothelial cells,
21 these data suggest a proangiogenic role of YKL-40 in the development of exudative AMD and could at least partly verify a protective role for estrogen replacement therapy.
12 13 A direct effect of YKL-40 on angiogenesis in other in vitro and in vivo models, such as corneal pellets or aortic rings, has yet to be evaluated. If a similar regulation were to be demonstrated in the synovium, our results could also provide a putative explanation for the protective effect of estrogens in rheumatoid arthritis, because YKL-40 has been proposed as a candidate autoantigen in this disease,
23 and its serum levels are related to disease activity.
25
It is of interest that, in our model of OVX mice, only a limited number of genes were affected by estrogen therapy in the retina. This could be explained by the pooling of samples for cDNA array, which selected only for strong and constant differences of gene expression and minimized individual variations. In particular, the expression of cathepsin D, an aspartic protease highly expressed in human retinal pigment epithelial lysosomes
40 with transcription classically regulated by estrogens in breast cancer,
41 was not influenced in vivo in the murine retina. Experimental impairment of cathepsin D results in accumulation of rod outer segment debris in the RPE and as been suggested as a murine model of dry AMD.
42 A similar discrepancy has been reported for endometrial cells and attributed to differential tissue-dependent regulation.
43 Individual variations in the level of mRNA expression for a specific gene are evident from the confirmation phase of our study, especially in the OVX untreated group. There was up to a 15-fold modulation of VEGF-A expression between mice of the same group. This could be explained by variations in very low level E2 concentrations (picomolar range), which have been demonstrated to enhance gene expression in different models.
44 It is also known that for some genes, the mechanism of regulation by estrogens is posttranscriptional rather than at the transcriptional level. This was indeed demonstrated for hepatic apolipoprotein E.
45 Finally, because the levels of ER-α and -β mRNA were weak in murine posterior segment, we cannot exclude the possibility that HRT could modify other genes in the human retina, in which these receptors are expressed at a higher level.
2
This study is obviously a preliminary phase in the understanding of the potential influences of HRT on AMD, and it would be of great interest to evaluate by cDNA microarray HRT-treated and untreated patients, as well as to compare intact and AMD tissue specimens. Our data nevertheless identify for the first time the YKL-40 gene as a potential mediator of estrogens effects both in the normal eye and during the development of CNV.