Role of granzyme B in the aging retina

该项目的目标是确定一个重要的酶家族，颗粒酶，在衰老的眼睛中的分布。颗粒酶是丝氨酸蛋白酶，我们的合作伙伴维达Therapeutics发现，颗粒酶在身体许多部位的细胞外蛋白和组织基质的异常降解中发挥重要作用。迄今为止，还没有关于颗粒酶在眼睛中的作用的研究，眼睛是一种含有重要的细胞外结构的器官，称为布鲁赫膜（BM）。随着眼睛年龄的增长，BM内的细胞外基质（ECM）经历急剧的重塑，推测会导致血眼屏障功能障碍。近年来的研究表明，视网膜外膜屏障功能的丧失导致视网膜细胞死亡、炎症反应、血管生成和血管通透性异常，本研究将为了解颗粒酶B在老年人眼内的细胞分布提供新的信息，有助于我们了解颗粒酶B在ECM重塑中的作用。假设随着年龄的增长，脉络膜组织中颗粒酶B的水平增加，导致BM受损，伴随着紧密连接蛋白的丢失，屏障功能失调，血管通透性增加，血管生成和促炎症。将评估来自死后人类供体（年龄范围为16岁至80岁）和野生型小鼠（C57 Bl/6 J，3至18月龄）的眼组织的颗粒酶B、细胞因子（IL 1 B; IL 1 a）、紧密连接蛋白（ZO-1、钙粘蛋白）和血管生成因子（VEGF、bFGF）的蛋白水平的细胞变化。免疫组织化学将确定层状分布，而基于抗体的多重检测将提供这些分子在视网膜中的定量值（pg/mL）。颗粒酶B基因敲除小鼠和颗粒酶B抑制剂治疗的老年小鼠的视网膜光学成像将评估颗粒酶B诱导的体内视网膜结构变化。主要优势包括合作伙伴专有颗粒酶B抑制剂的新目标领域。未来的经济和社会效益包括为加拿大慢性眼部炎症患者提供潜在的新治疗方法。

The goal of the project is to identify the distribution of an important family of enzymes, the Granzymes, in theaging eye. Granzymes are serine proteases that were shown by our partner, ViDA Therapeutics, to play animportant role in the abnormal degradation of extracellular proteins and tissue matrices in many parts of thebody. To date, there are no studies on the role of Granzymes in the eye, an organ that contains an importantextracellular structure called Bruch's Membrane (BM). As the eye ages, the extracellular matrix (ECM) withinBM undergoes dramatic remodelling, hypothesized to lead to a dysfunctional blood-eye barrier. Recent studieshave shown that loss of barrier function in the outer retina causes 1) retinal cell death, 2) pro-inflammatoryevents and 3) abnormal angiogenesis and vascular permeability.The research proposed will provide relevant and novel information about the cellular distribution ofGranzyme B in the aging eye that will allow us to understand Granzyme B's role in ECM remodeling. Thehypothesis is that with aging, levels of Granzyme B increase in the choroidal tissues, leading to a compromisedBM and concomitant loss of tight junctional proteins, dysregulated barrier function, increased vascularpermeability, angiogenesis and pro-inflammation. Eye tissues from postmortem human donors (ranging from16yr to 80yrs of age) and wild type mice (C57Bl/6J, 3 to 18 months of age) will be assessed for longitudinalchanges in protein levels of Granzyme B, cytokines (IL1b; IL1a), tight junctional proteins (ZO-1, cadherins),and angiogenic factors (VEGF, bFGF). Immunohistochemistry will identify the laminar distribution whileantibody-based multiplexes will provide quantitative values (pg/mL) of these molecules in the retina. Opticalimaging of the retina in Granzyme B knock-out mice and in aged mice treated with Granzyme B inhibitors willassess Granzyme B-induced changes in retinal structure in vivo. Key benefits include new target areas forpartner's proprietary Granzyme B inhibitors. Future economic and social benefits include potential newtreatments for Canadians suffering from chronic ocular inflammation.