Investigation and treatment of trabecular meshwork fibrosis using 3D glaucoma models

背景：开发安全有效的治疗纤维化的方法是青光眼患者的首要任务。这种眼病的特征是眼内压(IOP)升高，这是由于房水引流不力造成的。这在一定程度上是由于小梁网络(TM)1中细胞外基质沉积增加而导致的房水体液流出受阻所致。随着时间的推移，增加的压力可能会损害眼睛的结构，导致视力丧失。目前降低眼压的治疗策略包括无限期滴眼液，这可能会引起副作用，或者复杂的滤过手术。通过在手术时用丝裂霉素C等抗代谢物治疗组织，可以减少手术时的纤维化，但这与易发生渗漏、组织分解和感染的局部毒性有关。缺乏安全有效的抗纤维化治疗是一个重要的临床挑战。因此，确定药物开发的新靶点是很重要的。青光眼患者眼部的瘢痕形成和纤维化与小梁网的显著结构和功能重组有关，小梁网是眼内液体外流的主要抵抗部位。青光眼的病理生理机制尚未完全阐明，研究人员主要依靠活体啮齿动物模型3。这在一定程度上是由于缺乏有意义的组织工程和合适的体外模型。为了确定新的靶点和开发新的治疗方法，我们一直在与伯明翰和米德兰兹眼科中心合作，成功地从青光眼患者和非青光眼患者身上收集TM组织。因此，这一人类数据源不仅将为我们提供对疾病潜在机制的新见解，还将为我们提供新的靶向治疗策略的途径。目的：我们的目标是设计一种新的体外‘芯片上的器官’模型，以了解青光眼TM中发生的病理变化，并为青光眼的治疗寻找新的抗瘢痕化合物。我们将通过研究人类小梁网络(TM)中控制纤维化的机制以及与Schlemm管细胞的相互作用来实现这一点。我们将在3D模型中开发模拟人类小梁网/Schlemm管的共培养系统，然后使用该模型测试和筛选新的抗瘢痕治疗方法。此外，我们还将在取自外植体人体组织的体外实验中建立动态灌流模型(伦理和来源材料已就位)，以研究眼部瘢痕的压力诱导效应。使用对准的光学相干断层扫描(OCT)到‘芯片’模型，我们还将定义工程人类TM的机械性能的变化以及治疗的细胞和分子结果。培训结果：博士生将由一名眼生物学家(希尔博士)、一名生物材料科学家(格罗弗教授)和组织工程师(A El Haj教授)指导，并将得到临床眼科医生(马苏德先生，青光眼顾问)和细胞指导系统有限公司(Michael Jones博士)的工业支持。总体目标是减少使用我们的啮齿动物青光眼模型来评估新的抗瘢痕治疗的需要。在这个项目中，学生将接受有关人体组织处理(来自患者的样本)、细胞培养技术的培训，以开发重建胶原和弹性蛋白支架的3D体外模型(对TM进行建模)，并发展建立体外猪和人类模型的技能，以了解青光眼的病理并评估候选治疗方法。学生将学习常规的分子生物学技术(免疫细胞化学、免疫印迹、聚合酶链式反应)，以确定模型的特征并评估抗瘢痕治疗的效果，并有机会进行工业和国际安置。

Background: The development of safe and effective therapies to treat fibrosis is a major priority for patients with glaucoma. This ocular disease is characterised by elevated intraocular eye pressure (IOP), resulting from ineffective drainage of the aqueous humour. This in part is caused by the blockage of the aqueous humour outflow due to increased extracellular matrix deposition in the trabecular meshwork (TM)1. Over time the increased pressure can damage structures in the eye resulting in vision loss. Current therapeutic strategies to lower the IOP include indefinite eye-drops, which can cause side effects, or complex filtration surgery. Fibrosis at the time of surgery is reduced by treatment of tissue with anti-metabolites such as mitomycin C at the time of surgery, but this is associated with local toxicity predisposing to leaks, tissue breakdown and infections2. The lack of safe and effective anti-fibrotic treatments presents an important clinical challenge. It is therefore important to identify novel targets for drug development. Scarring and fibrosis of the eye in glaucoma is associated with marked structural and functional reorganisation of the trabecular meshwork, the main site of resistance to fluid outflow in the eye. The pathophysiology of glaucoma, has not yet been fully elucidated and investigators are mainly reliant on in vivo rodent models3. This in part has been due to the lack of meaningful tissue engineered and suitable ex vivo models. In order to identify novel targets and develop new treatments, we have been collaborating with Birmingham and Midlands Eye Centre to successfully collect TM tissue from patients with and without glaucoma. Therefore, this human data source will not only provide us with novel insights into the underlying mechanisms of the disease but also provide us with new pathways to target as a therapeutic strategy.Aims: Our aim is design a novel in vitro 'organ on a chip' model to understand the pathology which occurs in the TM in glaucoma and to identify novel anti-scarring compounds for the treatment of glaucoma. We will achieve this by investigating the mechanisms that control fibrosis in human trabecular meshwork (TM) and the interaction with Schlemm's Canal cells. We will develop co-culture systems in 3D models4 which mimic the human trabecular meshwork/Schlemm's canal and then use this model for testing and screening new anti-scarring treatments. In addition, we will develop dynamic perfusion models within ex vivo assays derived from explant human tissues (Ethics and sourced material in place) to investigate the pressure inducing effects of scarring in the eye. Using aligned optical coherence tomography (OCT) to the 'chip' models we will also define changes in mechanical properties of the engineered human TM alongside cell and molecular outcomes of treatments. Training outcomes: The PhD Candidate will be supervised by an ocular biologist (Dr Hill), a biomaterial scientist (Prof Grover) and tissue engineer (Prof A El Haj) and will have close input from a clinical ophthalmologist (Mr Masood, Glaucoma Consultant) and industrial support from the Cell Guidance Systems Ltd (Dr Michael Jones). The overall aim is to reduce the need to use our rodent glaucoma models to assess new anti-scarring treatments. Within this project the student will expect to receive training on human tissue processing (samples derived from patients), cell culture techniques for developing 3D in vitro models reconstructing collagen and elastin scaffolds (to model the TM) and to develop skills in setting up ex vivo porcine and human models for understanding glaucoma pathology and to assess candidate treatments. Students would learn routine molecular biology techniques (immunocytochemistry, western blots, PCR) in order to characterize the models and assess effects of anti-scarring treatments and have the opportunity for both industrial and international placements.