Role of endothelin receptors in glaucomatous optic neuropathy

描述（申请人提供）：青光眼是一种视神经病变，以视神经变性、视盘拔火罐、视网膜神经节细胞凋亡为特征，最终导致失明。眼压升高是原发性开角型青光眼（POAG）的重要危险因素。正常紧张性青光眼（NTG）患者也表现出视神经病变的迹象，即使他们有“正常”的IOPs。POAG和NTG患者分别在房水和循环中显示内皮素-1 （ET-1）升高，这是一种有效的血管活性肽。越来越多的证据表明，在青光眼实验模型中，ET-1参与介导视神经损伤。例如，连续给视神经球后区域注射小剂量的ET-1已被证明会在灵长类动物中产生视神经病变。然而，ET-1产生这些有害影响的机制尚不清楚。ET-1主要通过两类g蛋白偶联受体起作用，即ETA和ETB受体，它们主要分别与钙和一氧化氮信号通路相连。最近的观察表明，内皮素B受体在啮齿动物高IOP青光眼模型中表达上调。该建议的广泛目标是了解内皮素受体是否在青光眼动物模型中起神经退行性作用。要验证的总体假设是，大鼠IOP升高导致视网膜ETB受体表达增加，从而导致视网膜B神经节细胞死亡。以下具体目标将针对上述假设：1)使用免疫组织化学和受体结合研究相结合的方法测量IOP大鼠时间依赖性升高后大鼠视网膜中ETBB受体的表达。2)确定与野生型大鼠相比，ETB-B缺陷大鼠IOP升高后视网膜神经节细胞死亡是否减轻。这将通过在野生型和ETB-B缺陷大鼠IOP升高后计数可行的逆行标记视网膜神经节细胞来实现。3)通过启动子报告子和电泳迁移位移试验，了解视网膜神经节细胞中ETBB表达增加的分子机制，鉴定RGC-5细胞系中促进ETBB组成型和诱导型表达的顺式作用元件和反式作用因子。转录因子AP-1、C/EBP和SMAD 3/4的DNA结合活性也将在IOP升高的大鼠视网膜中进行研究，并与ETBB表达增加相关。这些研究将为青光眼视神经B病变中ETBB受体的表达和ETBB受体介导的介导视网膜神经节细胞凋亡的信号传导提供有价值的信息。从本研究中获得的信息可能有助于开发内皮素受体拮抗剂作为青光眼治疗中的神经保护剂。

DESCRIPTION (provided by applicant): Glaucoma is an optic neuropathy characterized by degeneration of the optic nerve, cupping of the optic disc and apoptosis of retinal ganglion cells, ultimately resulting in blindness. Elevated intraocular pressure (IOP) is a prominent risk factor as seen in primary open angle glaucoma (POAG). Normal tension glaucoma (NTG) patients also show sign of optic neuropathy even though they have "normal" IOPs. Both POAG and NTG patients show elevation of endothelin-1 (ET-1), a potent vasoactive peptide, in their aqueous humor and circulation respectively. Increasing evidence points to an involvement of ET-1 in mediating optic nerve damage in experimental models of glaucoma. For instance, continuous administration of small doses of ET-1 to the retrobulbar region of the optic nerve has been shown to produce optic neuropathy in primates. However, the mechanism by which ET-1 produces these deleterious effects are not understood. ET-1 acts through mainly two classes of G-protein coupled receptors, namely, the ETA and ETB receptors, which are B predominantly linked to the calcium and nitric oxide signaling pathways respectively. Recent observations indicate that there is an upregulation of endothelin B receptor expression in a rodent elevated IOP model of glaucoma. The broad goals of the proposal are to understand if endothelin receptors play a neurodegenerative role in an animal model of glaucoma. The overall hypothesis to be tested is that IOP elevation in rats produces increased ETB receptor expression in the retina which contributes to retinal B ganglion cell death. The following specific aims will be directed to addressing the above hypothesis: 1) Measure ETBB receptor expression in rat retinas following a time-dependent elevation of IOP rats using a combination of immunohistochemical and receptor binding studies. 2) Determine if retinal ganglion cell death following IOP elevation, is attenuated in ETB-B deficient rats, compared to wild type rats. This will be carried out by counting viable retrogradely labeled retinal ganglion cells following IOP elevation in wild type and ETB-B deficient rats. 3) Understand molecular mechanisms contributing to increase in ETBB expression in retinal ganglion cells by using promoter-reporter and electrophoretic mobility shift assays to identify cis-acting elements and trans-acting factors contributing to constitutive and inducible ETBB expression in the RGC-5 cell line. The DNA binding activity of transcription factors AP-1, C/EBP and SMAD 3/4 will also be studied in retinas of rats with elevated IOP and correlated with increased ETBB expression. These studies will provide valuable information on expression of the ETB receptor during glaucomatous optic B neuropathy and ETBB receptor mediated signaling that mediates apoptotic cell death of retinal ganglion cells. The information gained from this study could be useful in developing endothelin receptor antagonists as neuroprotective agents in glaucoma treatment. PUBLIC HEALTH RELEVANCE: The proposed study will determine if endothelin receptors produce some of the nerve damage seen in glaucoma. Understanding how endothelin receptors are damaging to nerve cells, will provide valuable information to block these receptors and thereby provide protection to nerve cells in the eye from further damage. This could lead to development of neuroprotection drugs as additional treatments to effectively treat glaucoma.