Differential proteomes in the vitreous of mice with a chronic hypoxic response in photoreceptors

Reduced oxygenation of photoreceptors and the RPE in the ageing eye may be a risk factor for the development of both neovascular and dry AMD. Chronic activation of the molecular response to hypoxia in RPE or photoreceptors leads to retinal degeneration that depends on hypoxia inducible transcription factors. For approaching the identification of accessible markers characteristic for photoreceptors with an activated hypoxic response, we used a proteomics approach to determine the protein composition of the vitreous humor in mice. To discriminate between rod and cone-specific effects we used genetically modified mice that had the hypoxic response activated specifically in rods of a rod-dominant retina (rodΔVhl) or a genetically engineered all-cone retina (coneΔVhl). For comparison, we used wild-type mice exposed for 6 hours to acute hypoxia. We identified 1,357 unique proteins in the vitreous of mice after acute hypoxia, 1,624 in rodΔVhl and 1,895 in coneΔVhl mice. Of these, 1,043 proteins were common to all three types of mice. Of the identified proteins, 257 were significantly regulated by a factor of 1.5 or more in hypoxic mice, 258 in rodΔVhl and 356 in coneΔVhl mice in at least one of the three analyzed time points. Only 51 of the significantly regulated proteins were common to the vitreous of rodΔVhl and coneΔVhl mice, suggesting different consequences of the activated hypoxic response for rods and cones. Guanylate binding protein 2 (GBP2) was found at increased levels in the vitreous of both rodΔVhl and coneΔVhl mice at all time points tested. This was also reflected by increased gene expression in the retina. Although retinal expression of the AMD-associated gene alpha-2 macroglobulin (A2M) appeared increased in both types of mice, the protein was only found elevated in the vitreous of rodΔVhl mice. Other proteins found increased included Serpina3n, synaptosome associated protein 25 (SNAP25) and others. The distinct protein compositions present at early and late time points, suggest a well-regulated process in our models. We hypothesize that some of the proteins identified at early time points may potentially be used as markers for the chronic hypoxic response of photoreceptors.

衰老眼球内光感受器与视网膜色素上皮（RPE，retinal pigment epithelium）的氧合水平降低，可能是新生血管性与干性年龄相关性黄斑变性（AMD，age-related macular degeneration）共同的发病风险因素。视网膜色素上皮或光感受器内缺氧分子应答的慢性激活，会依赖缺氧诱导转录因子引发视网膜变性。为鉴定具备激活型缺氧应答的光感受器的特异性可及标志物，我们采用蛋白质组学方法分析了小鼠玻璃体的蛋白组成。为区分视杆细胞与视锥细胞的特异性效应，我们使用了两类基因工程小鼠：一类在视杆主导型视网膜的视杆细胞中特异性激活缺氧应答（rodΔVhl），另一类为全视锥细胞视网膜模型（coneΔVhl）。作为对照，我们将野生型小鼠暴露于急性缺氧环境中6小时。我们在急性缺氧处理小鼠的玻璃体中鉴定出1357种独特蛋白，rodΔVhl小鼠中为1624种，coneΔVhl小鼠中为1895种。其中1043种蛋白在三类小鼠中均存在。在鉴定出的蛋白中，在至少三个分析时间点中的任意一个，缺氧小鼠体内有257种蛋白的表达变化幅度达到1.5倍及以上，rodΔVhl小鼠中为258种，coneΔVhl小鼠中为356种。仅51种显著调控的蛋白在rodΔVhl与coneΔVhl小鼠的玻璃体中共有，这表明视杆细胞与视锥细胞的缺氧应答激活会引发不同的生物学效应。鸟苷酸结合蛋白2（GBP2，Guanylate binding protein 2）在所有检测时间点的rodΔVhl与coneΔVhl小鼠玻璃体中均呈现表达水平升高。这一现象在视网膜的基因表达水平中也得到了印证。尽管与年龄相关性黄斑变性相关的α2巨球蛋白（A2M，alpha-2 macroglobulin）在两类小鼠的视网膜中表达均有所升高，但该蛋白仅在rodΔVhl小鼠的玻璃体中被检测到含量上调。其他被发现表达上调的蛋白包括丝氨酸蛋白酶抑制剂A3n（Serpina3n）、突触体相关蛋白25（SNAP25，synaptosome associated protein 25）等。早期与晚期时间点存在显著差异的蛋白组成表明，我们的模型中存在调控严谨的生物学过程。我们推测，早期时间点鉴定出的部分蛋白或可作为光感受器慢性缺氧应答的潜在标志物。